WO2020130077A1 - Composition for removing pluripotent stem cells and method of removing pluripotent stem cells - Google Patents

Composition for removing pluripotent stem cells and method of removing pluripotent stem cells Download PDF

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WO2020130077A1
WO2020130077A1 PCT/JP2019/049837 JP2019049837W WO2020130077A1 WO 2020130077 A1 WO2020130077 A1 WO 2020130077A1 JP 2019049837 W JP2019049837 W JP 2019049837W WO 2020130077 A1 WO2020130077 A1 WO 2020130077A1
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cells
stem cells
pluripotent stem
brq
mouse
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PCT/JP2019/049837
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French (fr)
Japanese (ja)
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亨 近藤
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国立大学法人北海道大学
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Priority to US17/414,511 priority Critical patent/US20220364065A1/en
Priority to EP19898801.6A priority patent/EP3901252A4/en
Priority to JP2020561509A priority patent/JP7409670B2/en
Publication of WO2020130077A1 publication Critical patent/WO2020130077A1/en

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Definitions

  • the present invention relates to a composition for removing pluripotent stem cells and a method for removing pluripotent stem cells. More specifically, the present invention relates to a composition and method for removing undifferentiated pluripotent stem cells remaining in a cell group in which differentiation is induced from pluripotent stem cells.
  • Pluripotent stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) have the ability to differentiate into all the cells that make up the living body, and therefore they are used as the key to the realization of regenerative medicine. Is expected. In addition, until now, various methods for inducing differentiation from these pluripotent stem cells to specific functional cells for transplantation required for treatment have been established.
  • ES cells embryonic stem cells
  • iPS cells induced pluripotent stem cells
  • Non-Patent Documents 1 to 5 various methods for removing pluripotent stem cells have been developed to solve this problem.
  • the present invention has been made in view of the above problems of the prior art, and finds a compound that brings cytotoxicity to undifferentiated pluripotent stem cells, while having little toxicity to other cells.
  • the purpose is to Furthermore, it is an object of the present invention to provide a composition for removing pluripotent stem cells containing the compound as an active ingredient, and a method for removing pluripotent stem cells using the compound.
  • DHODH dihydroorotate dehydrogenase
  • pluripotent stem cells by treating pluripotent stem cells with a DHODH inhibitor and then transplanting them into a mouse, or by administering a DHODH inhibitor to a mouse into which a pluripotent stem cells are transplanted, a particular side effect is brought about in the mouse. It was also confirmed that the tumor formation from pluripotent stem cells could be suppressed without the above.
  • DHODH is an oxidoreductase that catalyzes the fourth chemical reaction of de novo synthesis of pyrimidine. It has already been clarified that the DHODH inhibitor suppresses the proliferation of T cells and B cells and exerts an immunosuppressive effect by inhibiting the synthesis thereof, and the drug is due to such an effect, rheumatoid arthritis and the like. Is used for the treatment of autoimmune diseases of. Furthermore, DHODH inhibitors exert a therapeutic effect on leukemia (acute myeloid leukemia, chronic myelogenous leukemia) by releasing differentiation inhibition in cancer stem cells or by enhancing p53 synthesis in the same cells. It has also been reported (Non-patent documents 6 and 7).
  • the present inventor revealed for the first time that the drug can remove pluripotent stem cells, as described above. ..
  • somatic stem cells neural stem cells, etc.
  • somatic stem cells which are inferior to pluripotent stem cells but have pluripotency and self-proliferative ability and are also classified as stem cells
  • a DHODH inhibitor is The present inventors have found that they do not show significant cytotoxicity, and have completed the present invention.
  • the present invention relates to a composition for removing pluripotent stem cells and a method for removing pluripotent stem cells using a DHODH inhibitor, and more specifically provides the following.
  • the pluripotent stem cell is at least one pluripotent stem cell selected from the group consisting of embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells) and embryonic tumor cells (EC cells)
  • ES cells embryonic stem cells
  • iPS cells induced pluripotent stem cells
  • EC cells embryonic tumor cells
  • a method for removing residual undifferentiated pluripotent stem cells from the cell group which comprises a step of contacting a cell group differentiated from the pluripotent stem cells with a dihydroorotate dehydrogenase inhibitor.
  • the pluripotent stem cell is at least one pluripotent stem cell selected from the group consisting of embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells) and embryonic tumor cells (EC cells)
  • ES cells embryonic stem cells
  • iPS cells induced pluripotent stem cells
  • EC cells embryonic tumor cells
  • ⁇ 7> The method according to ⁇ 5> or ⁇ 6>, wherein the dihydroorotic acid dehydrogenase inhibitor is brequinar.
  • the cell group is a cell group containing somatic stem cells differentiated from the pluripotent stem cells.
  • undifferentiated pluripotent stem cells can be removed without causing significant cytotoxicity to differentiated cells. That is, according to the present invention, it becomes possible to remove the undifferentiated pluripotent stem cells remaining in the cell group differentiated from the pluripotent stem cells.
  • FIG. 1 is a graph showing the survival rate of mouse embryonic stem (ES) cells after culturing for 3 days in the presence of a dihydroorotic acid dehydrogenase (DHODH) inhibitor.
  • DHODH dihydroorotic acid dehydrogenase
  • 3 is a fluorescence micrograph showing the results of mixed culture of mouse neural stem cells and mouse ES cells for 3 days in the presence of BRQ.
  • control indicates the culture result in the absence of BRQ.
  • the upper three photographs show the results of detection of a neural stem cell marker (Nestin) by immunostaining.
  • the middle three photographs show the results of detecting pluripotent stem cell markers (Nanog) by immunostaining.
  • the bottom three photographs are photographs in which the results of detecting Nestin and Nanog by immunostaining and the results of counterstaining the cell nuclei with DAPI are superimposed.
  • FIG. 3 is a fluorescence micrograph showing the results of mixed culture of mouse neural stem cells and mouse iPS cells for 3 days in the presence of BRQ.
  • control indicates the culture result in the absence of BRQ.
  • the upper three photographs show the results of detecting a pluripotent stem cell marker (Nanog) by immunostaining.
  • the middle three photographs show the results of detecting a neural stem cell marker (Nestin) by immunostaining.
  • the bottom three photographs are photographs in which the results of detecting Nestin and Nanog by immunostaining and the results of counterstaining the cell nuclei with DAPI are superimposed.
  • FIG. 9 is a graph showing the survival rate of mouse iPS cells when various ribonucleosides (uridine, adenosine, guanosine or cytidine) were added in the presence of 10 ⁇ M BRQ.
  • 6 is a graph showing the survival rate of mouse ES cells when various ribonucleosides (uridine, adenosine, guanosine or cytidine) were added in the presence of 10 ⁇ M BRQ.
  • FIG. 2 is a graph showing the survival rate of mouse iPS cells when various ribonucleosides (uridine diphosphate (UDP) or uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc)) were added in the presence of 10 ⁇ M BRQ.
  • 6 is a graph showing the survival rate of mouse ES cells when various ribonucleosides (UDP or UDP-GlcNAc) were added in the presence of 10 ⁇ M BRQ.
  • FIG. 6 is a graph showing the dose-dependent effect of nucleotide diphosphate on BRQ-dependent cytotoxicity in pluripotent stem cells.
  • ESC indicates the effect on mouse ES cells
  • iPSC indicates the effect on mouse iPS cells.
  • the circles show the dose-dependent effects of UDP, the diamonds of CDP, the squares of ADP, and the triangles of GDP. Error bars indicate ⁇ SD (standard deviation). Statistical significance was determined by t-test. ***P ⁇ 0.01, ***p ⁇ 0.001. It is a graph which shows the ratio of BrdU positive cell in a pluripotent stem cell in presence of BRQ.
  • ESC indicates the ratio in mouse ES cells
  • iPSC indicates the ratio in mouse iPS cell cells.
  • N and BRQ indicate the absence and presence of BRQ in the culture medium of pluripotent stem cells, respectively. It is a graph which shows the ratio of the Casp3 positive cell in a pluripotent stem cell in the presence of BRQ.
  • ESC indicates the ratio in mouse ES cells
  • iPSC indicates the ratio in mouse iPS cell cells.
  • N and BRQ indicate the absence and presence of BRQ in the culture medium of pluripotent stem cells, respectively. It is a photograph which shows the result of having analyzed the knockdown efficiency by a DHODH expression vector by western blotting.
  • the FLAG-tagged mouse DHODH expression vector was transferred to a Cos7 cell together with a vector encoding a control shRNA (“C” in the figure) or a vector encoding shRNA1 to 3 for DHODH (“DHODH sh1, sh2, sh3” in the figure). Introduced. Two days after the gene transfer, the cell extract was collected and analyzed by Western blotting using anti-FLAG antibody and anti-GAPDH antibody (loading control). It is a graph which shows the ratio of Ki67 positive cell in a control shRNA expression pluripotent stem cell or a DHODHshRNA expression pluripotent stem cell.
  • ESC and iPSC indicate the above-mentioned proportions in mouse ES cells and mouse iPS cells, respectively. Error bars indicate ⁇ SD. Statistical significance was determined by t-test. *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001. It is a graph which shows the ratio of a Casp3 positive cell in a control shRNA expression pluripotent stem cell or a DHODHshRNA expression pluripotent stem cell. In the figure, “ESC” and “iPSC” indicate the above-mentioned proportions in mouse ES cells and mouse iPS cells, respectively. Error bars indicate ⁇ SD. Statistical significance was determined by t-test.
  • FIG. 3 is a fluorescence micrograph showing representative results obtained by observing control shRNA-expressing pluripotent stem cells and dhodhsh-expressing pluripotent stem cells by immunostaining for GFP and Ki67.
  • ESC and “iPSC” indicate the results of observing mouse ES cells and mouse iPS cells, respectively.
  • Each of the three photographs in the first and third rows shows the results of detecting Ki67 (red) expression and nuclei (blue, counterstaining with DAPI) in mouse ES cells and mouse iPS cells.
  • FIG. 3 is a fluorescence micrograph showing representative results obtained by observing control shRNA-expressing pluripotent stem cells and dhodhsh-expressing pluripotent stem cells by immunostaining for GFP and Casp3.
  • ESC and “iPSC” indicate the results of observing mouse ES cells and mouse iPS cells, respectively.
  • the three photographs in the first and third rows show the results of detecting the expression of Casp3 (red) and the nucleus (blue, counterstaining with DAPI) of mouse ES cells and mouse iPS cells.
  • the three photographs in the second and fourth rows show the results of detecting the expression of GFP (green) and Casp3 (red) in mouse ES cells and mouse iPS cells.
  • the scale bar shows 100 ⁇ m. After culturing for 24 hours in the presence of 10 ⁇ M BRQ, it is a photograph showing the results of observing each cell with a fluorescence microscope.
  • “NSC”, “iPS” and “ES” indicate the results of observing mouse neural stem cells, mouse iPS cells and mouse ES cells.
  • BRQ indicates the result of culturing in the presence of 10 ⁇ M BRQ
  • DMSO indicates the result of culturing in the absence of BRQ.
  • the upper 6 photographs show the results of immunostaining Sox2, and the lower 6 photographs are the photographs in which the results of the immunostaining of Sox2 and the results of counterstaining the cell nuclei with DAPI are superimposed.
  • Arrows indicate Sox2-negative cells. Each numerical value shows the ratio (%) of Sox2-positive cells to DAPI-positive cells. Scale bar represents 100 ⁇ m.
  • 3 is a graph showing the ratio (%) of Sox2-positive cells in each cell after culturing for 24 hours in the presence of 10 ⁇ M BRQ.
  • NSC NSC
  • iPS ES
  • ES ES
  • BRQ indicates the result of culturing in the presence of 10 ⁇ M BRQ
  • Const indicates the result of culturing in the absence of BRQ.
  • ** indicates that P value ⁇ 0.01.
  • 3 is a graph showing the expression levels of pluripotent stem cell marker genes (Sox2, Oct4 and Nanog) in each cell after culturing in the presence of 10 ⁇ M BRQ for 1 day or 2 days.
  • BRQ indicates the result of culturing in the presence of 10 ⁇ M BRQ
  • DMSO indicates the result of culturing in the absence of BRQ.
  • the vertical axis of each graph shows a relative value when the expression level of each gene in "DMSO” is 100.
  • ES and “iPS” indicate the results of observing mouse ES cells and mouse iPS cells.
  • BRQ indicates the result of culturing in the presence of 10 ⁇ M BRQ
  • cont indicates the result of culturing in the absence of BRQ.
  • the upper four photographs show the results of immunostaining for Nanog, and the lower four photographs are the photographs in which the results of immunostaining of Nanog and the results of counterstaining cell nuclei with DAPI are superimposed.
  • Arrows indicate cells in which Nanog is spread and distributed in the cytoplasm (cells in which Nanog is excluded from the nucleus).
  • Pluripotent stem cells were cultured for 2 days in the presence of DMSO, ZVAD (100 nM), BRQ (10 ⁇ M) or BRQ and ZVAD (BRQ:10 ⁇ M, ZVAD:100 nM), and immunolabeled with Nanog and Oct4. It is a fluorescence micrograph which shows a result.
  • ESC and “iPSC” indicate the results of observing mouse ES cells and mouse iPS cells.
  • the numerical values in the figure show the ratio of the number of cells positive for each nuclear PSC marker in all cells.
  • the upper 8 photographs show the results of immunostaining Oct4 (green), the middle 8 photographs show the results of immunostaining Nanog (red), and the lower 8 photographs show the results of immunostaining Oct4 and The result of superimposing the result of counter-staining the cell nucleus (blue) with DAPI on the result of immunostaining Nanog is shown.
  • the scale bar shows 50 ⁇ m.
  • Pluripotent stem cells were cultured in the presence of DMSO, BRQ (10 ⁇ M) or BRQ and LMB (BRQ:10 ⁇ M, LMB: 0.15 nM) for 2 days, and Nanog (red) and Oct4 (green) were cultured. It is a fluorescence micrograph which shows the result of immunolabeling.
  • ESC and "iPSC” indicate the results of observing mouse ES cells and mouse iPS cells, respectively.
  • the numerical values in the figure show the ratio of the number of cells positive for each nuclear PSC marker in all cells.
  • the upper 6 photographs show the results of immunostaining Oct4 (green), the middle 6 photographs show the results of immunostaining Nanog (red), and the lower 6 photographs show the results of immunostaining Oct4 and The results of immunostaining Nanog with the results of counterstaining the cell nuclei (blue) with DAPI are shown.
  • the scale bar shows 100 ⁇ m. Representative photographs of teratomas formed from pluripotent stem cells pretreated with DMSO or BRQ are shown. In the figure, “ESC” and “iPSC” indicate the results of observing teratomas formed from mouse ES cells and mouse iPS cells, respectively. Scale bar indicates 1 cm.
  • FIG. 6 is a graph showing the volume of tumors formed from pluripotent stem cells pretreated with DMSO or BRQ.
  • ESC and “iPSC” indicate the volume of tumor formed from mouse ES cells and mouse iPS cells, respectively. Error bars indicate ⁇ SD. Statistical significance was determined by t-test. ***P ⁇ 0.01, ***P ⁇ 0.001. Representative photographs of teratomas formed from pluripotent stem cells in mice treated with DMSO or BRQ are shown.
  • “ESC” and “iPSC” indicate the results of observing teratomas formed from mouse ES cells and mouse iPS cells, respectively. Scale bar indicates 1 cm.
  • 6 is a graph showing the tumor volume of teratomas formed from pluripotent stem cells in mice treated with DMSO or BRQ.
  • “ESC” and “iPSC” indicate tumor volumes formed from mouse ES cells and mouse iPS cells, respectively. Error bars indicate ⁇ SD.
  • Statistical significance was determined by t-test. ***P ⁇ 0.01, ***P ⁇ 0.001.
  • 6 is a graph showing the proportion of Ki67-positive cells in teratomas formed from pluripotent stem cells in mice treated with DMSO or BRQ.
  • "ESC” and “iPSC” indicate the above-mentioned proportions in teratoma formed from mouse ES cells and mouse iPS cells, respectively. Error bars indicate ⁇ SD.
  • ESC and “iPSC” indicate the results of observing teratomas formed from mouse ES cells and mouse iPS cells, respectively. Nuclei were detected by counterstaining with DAPI (blue). The scale bar shows 100 ⁇ m.
  • pluripotent stem cells can be cytotoxicized and removed. Revealed.
  • somatic stem cells obtained by inducing differentiation of pluripotent stem cells or differentiated cells such as somatic cells, even if DHODH is inhibited, significant cytotoxicity does not occur. ..
  • the present invention provides a composition and a method for removing undifferentiated pluripotent stem cells remaining in a cell population differentiated from pluripotent stem cells using a DHODH inhibitor.
  • dihydroorotic acid dehydrogenase means an enzyme that catalyzes the oxidation of dihydroorotic acid to orotic acid, which is the fourth reaction in the de novo synthetic pathway of pyrimidine, and includes, for example, dihydroorotic acid dehydrogenase (fumaric acid). ) (EC number: 1.3.98.1), dihydroorotic acid dehydrogenase (NAD+) (EC number: 1.3.1.14), dihydroorotic acid dehydrogenase (NADP+) (EC number: 1.3.1.15). ), and dihydroorotic acid dehydrogenase (quinone) (EC number: 1.3.5.2).
  • DHODH dihydroorotic acid dehydrogenase
  • DHODH inhibitor means a compound having an activity of inhibiting the catalytic reaction.
  • the “inhibition” in the present invention includes not only complete inhibition of activity and the like but also partial inhibition (suppression).
  • Examples of the “DHODH inhibitor” according to the present invention include brequinar (BRQ, 6-fluoro-2-(2′-fluoro-1,1′-biphenyl-4-yl), as shown in Examples below.
  • -3-methyl-4-quinoline-carboxylic acid sodium salt leflunomide (leflunomide, 5-methyl-N-[4-(trifluoromethyl)phenyl]-isoxazole-4-carboxamide), teriflunomide, (2Z )-2-Cyano-3-hydroxy-N-[4-(trifluoromethyl)phenyl]but-2-enamide), Vidofludimus, 2-(3-Fluoro-3′-methoxybiphenyl-4-ylcarbamoyl) )-Cyclopent-1-enecarboxylic acid
  • the “DHODH inhibitor” according to the present invention is preferably a compound having a 50% inhibitory concentration (IC 50 ) for human-derived DHODH of 100 nM or less, more preferably a compound having an IC 50 of 50 nM or less, and an IC 50 of 20 nM.
  • IC 50 50% inhibitory concentration
  • the following compounds are more preferable. Examples of such compounds include the following compounds (the structure of each compound and the IC 50 for human-derived DHODH are shown below).
  • the enzyme activity of DHODH can be determined by those skilled in the art by, for example, a dye reduction assay using dichloroindophenol (DCIP).
  • DCIP dichloroindophenol
  • the activity of DHODH can be determined using the decrease in the absorbance at the wavelength as an index.
  • the IC 50 for DHODH is, for example, that DOHDH derived from human, a substrate (dihydroorotic acid), an electron acceptor (for example, CoQ), and an enzyme reaction solution containing a buffer have various concentrations of a test compound (DHODH inhibition). Agent) and perform the DCIP assay (see WO 2008/077639, Biochem J. 1998 Dec 1;336(Pt2):299-303).
  • the “DHODH inhibitor” according to the present invention also includes a pharmacologically acceptable salt, hydrate or solvate as long as it has the inhibitory activity.
  • a pharmacologically acceptable salt is not particularly limited and can be appropriately selected according to each structure of the drug, and examples thereof include acid addition salts (hydrochloride, sulfate, hydrobromide). , Nitrates, hydrogensulfates, phosphates, acetates, etc.) and base addition salts (sodium salts, potassium salts, zinc salts, calcium salts, etc.).
  • the hydrate or solvate is not particularly limited, and examples thereof include those in which 0.1 to 10 molecules of water or solvent are added to 1 molecule of DHODH inhibitor or its salt.
  • the “DHODH inhibitor” includes all isomers such as tautomers, geometric isomers, optical isomers based on asymmetric carbons, stereoisomers and the like, as long as it has the inhibitory activity. Includes isomer mixtures.
  • DHODH inhibitors undergo metabolism in vivo such as oxidation, reduction, hydrolysis, amination, deamination, hydroxylation, phosphorylation, dehydroxylation, alkylation, dealkylation, conjugation, etc., and still inhibit the metabolism.
  • the present invention also includes compounds that exhibit activity, and the present invention also includes compounds that are metabolized in vivo by oxidation, reduction, hydrolysis and the like to produce DHODH inhibitors.
  • DHODH inhibitor many compounds have been developed and are on the market as described above, so they can be obtained by purchasing. Many reports have been made on methods for producing these compounds even if they are not commercially available. Therefore, a person skilled in the art can also appropriately prepare according to the manufacturing method.
  • the “pluripotent stem cell” may be any cell having pluripotency and self-renewal ability, and examples thereof include embryonic stem cells (ES cells), embryonal tumor cells (EC cells), and epiblasts. Stem cells (EpiS cells), embryonic germ cells (EG cells), pluripotent germ cells (mGS cells), MUSE cells (Kuroda Y. et al., Proc. Natl. Acad. Sci. USA, 2010) , Vol. 107, No. 19, pp. 8639-8643). Furthermore, "pluripotent stem cells” also include cells derived from somatic cells collected from a living body so as to have artificial pluripotency, such as induced pluripotent stem cells (iPS cells). Be done.
  • iPS cells induced pluripotent stem cells
  • the origin of these cells is not particularly limited, and humans and non-human animals (for example, rodents such as mice and rats, cows, horses, pigs, sheep, monkeys, dogs, mammals such as cats, chickens, etc.) Birds). Furthermore, in the case of aiming at the regenerative medicine described below, pluripotent stem cells derived from the target to which the differentiated cells described below are transplanted are desirable from the viewpoint of suppressing immune rejection.
  • undifferentiated pluripotent stem cells include, in addition to the pluripotent stem cells that have not been differentiated and maintain pluripotency, cells in which pluripotent stem cells have turned into tumors (for example, Teratoma, tetracarcinoma) are also included.
  • the “cell group differentiated from pluripotent stem cells” means a population of cells including any differentiated cells derived from the above pluripotent stem cells.
  • Such a cell group may include a single type of differentiated cell or may include a plurality of types of differentiated cells. Further, the form may be a tissue or an organ composed of these cells.
  • differentiated cells include not only cells that have completely lost pluripotency (terminally differentiated cells, such as somatic cells and germ cells) but also cells that have partially lost pluripotency (eg, progenitor cells). Cells, somatic stem cells) are also included.
  • somatic stem cells are cells also called adult stem cells and tissue stem cells, and include, for example, neural stem cells, satellite cells, hematopoietic stem cells (bone marrow stem cells), mesenchymal stem cells, intestinal stem cells, hair follicle stem cells, mammary stem cells, Examples include endothelial stem cells, olfactory mucosa stem cells, neural crest stem cells, and testis cells.
  • Induction of differentiation of pluripotent stem cells into “differentiated cells” is usually carried out through induction of differentiation into germ layers (ectodermal, mesoderm, endoderm) in which each differentiated cell develops.
  • a known method using a low-molecular compound, a protein or the like (differentiation inducing factor) suitable for inducing differentiation into each germ layer cell can be appropriately selected and performed.
  • ectodermal cells such as nerve cells can be induced to differentiate by culturing in the presence of a BMP inhibitor, TGF ⁇ and an activin inhibitor (Nat Biotechnol. 2009 Mar; 27(3):275. -80).
  • Differentiation can be induced into mesodermal cells such as chondrocytes by culturing pluripotent stem cells in the presence of Wnt and activin, and then culturing them in the presence of BMP4 ( Development.2008 Sep;135(17):2969-79).
  • endodermal cells such as pancreatic cells and intestinal cells can be induced to differentiate by culturing pluripotent stem cells in the presence of activin and then culturing them in the presence of BMP4 (for example, , Diabetes 2010 Sep; 59(9):2094-2101, Nature. 2011 Feb 3; 470(7332): 105-9).
  • “removal” of undifferentiated pluripotent stem cells remaining in the above-mentioned cell group means not only complete removal of the cells but also reduction of the number of the cells or the ratio in the cell group. To do.
  • the removal may be performed in vitro, or may be performed in vivo or ex vivo.
  • Embodiments of the present invention include a composition containing a DHODH inhibitor as an active ingredient for removing undifferentiated pluripotent stem cells remaining in a cell group differentiated from pluripotent stem cells.
  • composition of the present invention may contain a physiologically acceptable carrier in addition to the above-mentioned DHODH inhibitor.
  • physiologically acceptable carriers include physiological isotonic solutions (physiological saline, culture medium, glucose and other isotonic solutions containing D-sorbitol, D-mannitol, sodium chloride, etc.). ), excipients, preservatives, stabilizers (human serum albumin, polyethylene glycol, etc.), binders, solubilizing agents, nonionic surfactants, buffers (phosphate buffer, sodium acetate buffer, etc.) , Preservatives and antioxidants.
  • composition of the present invention may be in the form of a reagent used for research purposes or medical purposes (for example, production of differentiated cells, particularly differentiated cells with reduced tumorigenicity risk). Further, as described below, it may be in the form of a pharmaceutical composition to be administered to a subject into which the differentiated cells have been transplanted.
  • the present invention also provides a kit for producing a differentiated cell, particularly a differentiated cell with reduced tumorigenicity risk.
  • a kit for producing a differentiated cell in addition to the composition, pluripotent stem cells, the above-mentioned differentiation-inducing factor, differentiation-inducing medium, incubator, and other materials necessary for inducing differentiation of pluripotent stem cells into desired cells are included. be able to.
  • a reagent for detecting the cell-specific marker molecule for example, an antibody specific to the marker molecule
  • the kit of the present invention also includes instructions indicating the method of using the composition of the present invention, the method of inducing differentiation and the like.
  • the embodiment of the present invention also includes a step of contacting a group of cells differentiated from pluripotent stem cells with a dihydroorotic acid dehydrogenase inhibitor, a method of removing undifferentiated pluripotent stem cells remaining from the group of cells Are listed.
  • the “contact” between the above-mentioned DHODH inhibitor and the cell group can be performed by adding the DHODH inhibitor to the medium for maintaining the cell group.
  • the concentration to be added at that time is not particularly limited and can be appropriately adjusted by those skilled in the art depending on the types of the pluripotent stem cells and cell groups of interest, the type of DHODH inhibitor to be used, etc. It is 0.1 to 1000 ⁇ M, preferably 1 to 100 ⁇ M, and more preferably 10 to 50 ⁇ M.
  • the method of the present invention can be rephrased as "a method for producing differentiated cells with reduced tumorigenicity risk, which comprises the step of contacting a cell group differentiated from pluripotent stem cells with a dihydroorotate dehydrogenase inhibitor"
  • the DHODH inhibitor and the cell group can be treated by administering the DHODH inhibitor to the subject. Contact can be made.
  • the dosage form is not particularly limited, and examples thereof include intravenous administration, intraarterial administration, intraperitoneal administration, subcutaneous administration, intradermal administration, respiratory tract administration, rectal administration and intramuscular administration, administration by infusion, local administration, and oral administration. It is appropriately selected depending on the type of cell group to be transplanted, the site of transplantation and the like.
  • the dose is appropriately adjusted depending on the type of subject, age, weight, symptoms and health condition, administration form, types of target pluripotent stem cells and cell groups, types of DHODH inhibitors used, and the like.
  • the dose per administration is usually 0.001 to 1000 mg/kg body weight, preferably 0.1 to 100 mg/kg body weight, and more preferably 1 to 50 mg/kg body weight.
  • the number of administrations per day is also not particularly limited, and may be adjusted appropriately in consideration of various factors as described above. Further, the subject may be continuously administered (eg, once a week).
  • the contact time between the DHODH inhibitor and the cell population for removing the undifferentiated pluripotent stem cells may be any time sufficient for the removal, and those skilled in the art can appropriately adjust the time. However, it is usually 2 to 7 days, preferably 2 to 5 days, and more preferably 2 to 3 days.
  • mice All experiments using mice were performed according to the protocol approved by the Hokkaido University Animal Experiment Committee. The mouse used was purchased from Hokudo Co., Ltd. Unless otherwise specified, chemicals and growth factors used were those purchased from Invitrogen and PeproTech, respectively.
  • mice Pluripotent stem cells
  • E14tg2a a cell line given by Mr. Shinji Masui (at the time of receiving the cells, enrolled in Kyoto University iPS Laboratory) was used.
  • a cell line iPS-Hep-FB/Ng/gfp-103c-1 purchased from RIKEN BioResource Research Center was used.
  • pluripotent stem cells were cultured by the method disclosed on the SIGMA website (https://www.sigmaaldrich.com/life-science/stem-cell-biology/system-cell-protocols.html). reference).
  • a culture medium (ESC culture medium) having the following composition was used.
  • the surface of a 100 mm culture dish (manufactured by Falcon) was coated with gelatin derived from bovine epidermis (manufactured by Sigma, product number: G9391) and kept at 37° C. for 2 hours or more. The placed one was used.
  • EC cells Human embryonal carcinoma (EC) cells (NT2) were obtained from the American Tissue Culture Collection (CRL-1973) and cultured in DMEM (Dulbecco's modified Eagle medium) containing 10% FBS.
  • DMEM Dulbecco's modified Eagle medium
  • mice Mouse neural stem cells (mNSCs) are described in Johe KK et al., Genes Dev. 1996, Vol. 10, No. 24, pp. 3129-3140, prepared from the telencephalon of mice (fetus 14.5 days), and cultured in NSC medium [bFGF (10 ng/ml), EGF (10 ng/ml). ), heparin (5 ⁇ M), GlutaMAX (manufactured by Gibco), penicillin, and streptomycin in DMEM/F12 medium (manufactured by Sigma)].
  • Mouse bone marrow-derived stromal cells were obtained from Riken BioResource Center (RBRC-RCB1127) and cultured in ⁇ MEM containing 10% FBS (Eagle minimum essential medium ⁇ modified type).
  • Mouse myoblasts (C2C12) were obtained from Riken BioResource Center (RBRC-RCB0987) and cultured in DMEM containing 10% FBS.
  • Astrocytes were prepared by culturing mNSC in the presence of DMEM containing 10% FBS for 3 days, and then maintained in the same medium.
  • MTT assay method 1000 cells were seeded in each well of a 96-well plate and DMSO alone was added to a medium (200 ⁇ L/well) to a final concentration of 0.1%, or each dihydroorotic acid dehydrogenase (DHODH) inhibitor The cells were cultured for 3 days in a medium (200 ⁇ L/well) supplemented with.
  • the culture medium used was a culture medium suitable for each of the cells described above.
  • a DHODH inhibitor As a DHODH inhibitor, Brequnar (BRQ, Cayman Chemical, CAS number: 96187-53-0), Leflunomide (Cayman Chemical, CAS number: 75706-12-6), Teriflunomide (Cayman45:Chamical 81ChemicalChemical number: 81:Chamman: 45-81-Chemical number: 45-81-Chemical number: 16-Chemical-Chemical number) ) Or Vidofludimus (Cayman, CAS number: 717824-30-1) was added to the medium. The concentration of each DHODH inhibitor added to the medium was a 2-fold dilution series starting from 200 ⁇ M.
  • a well containing only the medium was prepared, and as a control, a well containing a medium containing Triton X-100 (final concentration 0.2%) was added to the cells.
  • ES cells or iPS cells were incubated for 3 days in the presence of BRQ (10 ⁇ M) or in the presence of BRQ (10 ⁇ M) and various concentrations of ribonucleosides (up to 500 ⁇ M, all manufactured by Sigma), and the MTT described above was added. Cell viability was analyzed by assay.
  • the cells were washed once with PBS, added with 4% paraformaldehyde, and incubated at room temperature for 10 minutes for fixation. After replacing with PBS containing 0.3% Triton X100 and incubating at room temperature for 5 minutes, it was replaced with PBS containing 0.3% Triton X100 and 10% FCS (blocking solution) and incubated at room temperature for 30 minutes. The solution was replaced with a primary antibody solution diluted with a blocking solution, and incubated at room temperature for 2 hours. After washing three times with PBS, the secondary antibody diluted with the blocking solution and the DAPI solution were added, and the mixture was incubated at room temperature for 2 hours.
  • the cells were encapsulated with an anti-fading agent (manufactured by DAKO, code number: S3023) and observed under a microscope.
  • the cell nuclei were visualized by counterstaining with a DAPI solution (1 ⁇ g/mL, manufactured by Dojindo, product code: D523).
  • the fluorescence image was acquired with an AxioImager M1 microscope (Carl Zeiss).
  • Anti-Nestin mouse monoclonal antibody (BD, clone Rat401, diluted 1:200 for use), Anti-Nanog rabbit polyclonal antibody (manufactured by Wako pure chemical, code number: 018-27521, used by diluting 1:200), Anti-Sox2 rabbit polyclonal antibody (manufactured by StemCell Technology, diluted 1:500 for use), Anti-Oct4 mouse monoclonal antibody (Cell Signaling Tech, diluted 1:200 for use), Anti-GFP rat monoclonal antibody (manufactured by Nacalai Tesque, Inc., diluted 1:500), Anti-Ki67 rabbit monoclonal antibody (Thermo Fisher Scientific, manufactured at a dilution of 1:100), Anti-active Caspase3 rabbit polyclonal antibody (manufactured by StemCell Technology,
  • Alexa488-labeled anti-mouse IgG donkey polyclonal antibody (Thermo Fisher Scientific, catalog number: R37120, diluted 1:500 for use), Alexa488-labeled anti-rabbit IgG donkey polyclonal antibody (Thermo Fisher Scientific, catalog number: A-11008, diluted 1:500 for use), Alexa 594-labeled anti-rabbit IgG donkey polyclonal antibody (Thermo Fisher Scientific, catalog number: R37119, diluted 1:500 for use), Alexa 594-labeled anti-mouse IgG donkey polyclonal antibody (Thermo Fisher Scientific, catalog number: R37115, diluted 1:500 for use), Alexa488 labeled anti-rat IgG goat antibody (manufactured by Jackson ImmunoResearch, diluted 1:500 and used).
  • Anti-Oct4 mouse monoclonal antibody Cell Signaling Tech, diluted 1:200 for use
  • Anti-Nanog rabbit polyclonal antibody manufactured by Wako Pure Chemical, diluted 1:200 for use
  • Anti-Ki67 rabbit monoclonal antibody Thermo Fisher Scientific, manufactured at a dilution of 1:100
  • Anti- ⁇ III tubulin mouse monoclonal antibody Sigma, diluted 1:400 for use
  • Anti-smooth muscle actin rabbit polyclonal antibody manufactured by Proteintech, SMA, diluted 1:200 for use
  • Anti-alpha fetoprotein rabbit polyclonal antibody Proteintech, AFP, diluted 1:100 for use).
  • Alexa 594-labeled anti-mouse IgG donkey polyclonal antibody (Thermo Fisher Scientific, catalog number: R37115, diluted 1:500 for use), Alexa488-labeled anti-rabbit IgG goat antibody (Jackson ImmunoResearch, 1:500 dilution).
  • mice NSCs and mouse ES cells or mouse iPS cells were seeded, and cultured in the ESC culture medium for 3 days in the presence of 1 ⁇ M or 10 ⁇ M BRQ. And it analyzed by the above-mentioned immunostaining.
  • Sox2 gene Forward primer 5'-TGAAGAAGGAATAAGTACACCGCT-3' (SEQ ID NO: 1), Reverse primer 5'-TCCTGCATCATGCTGTAGCTG-3' (SEQ ID NO: 2), oct4 gene: Forward primer 5′-CTGAAGCAGAAGAGGATCACC-3′ (SEQ ID NO: 3), Reverse primer 5'-CCGCAGCTTACACATGTTCTT-3' (SEQ ID NO: 4), nanog gene: Forward primer 5'-CTGATTCTTCTACCAGTCCCCAA-3' (SEQ ID NO: 5), Reverse primer 5'-AGAGTTCTTGCATCTGCTGGA-3' (SEQ ID NO: 6), 18S ribosomal RNA gene: Forward primer 5′-CGGACAGGATTGACAGATTG-3′ (SEQ ID NO: 7), Reverse primer 5'-CAAATCGCTCCACCAACTAA-3' (SEQ ID NO: 8). The expression level of each target gene thus detected was normalized by the relative quantification method (delt
  • Vectors are based on Nishide K, et al., PLoS ONE. 2009; 4(8):e6869. And Takanaga H, et al., Stem Cells. 2009;27(1):165-174. It was constructed according to the description.
  • the full-length cDNA of mouse DHODH is KOD Plus-Ver. 2 polymerase (manufactured by Toyobo Co., Ltd.) was used and amplified from mouse NSC-derived cDNA according to the instruction manual.
  • a 5'primer (5'-AGAATTCAATGGGGGAACA-3' (SEQ ID NO: 9)
  • a 3'primer (5'-TTGGATTCTCTGCCGGTC-3' (SEQ ID NO: 10) were used for the amplification.
  • the amplified cDNA was inserted into the p3xFLAG CMV10 vector to prepare p3xFLAG CMV10-mDHODH.
  • the target sequences of sh1 and sh3 targeting mouse DHODH are 5'-GGCTAGCTGTCtCTCTCT-3' (SEQ ID NO: 11) and 5'-GGAAGCTGTGTCTCTCA-3' (SEQ ID NO: 12), respectively.
  • the target sequence of sh(egfp) used as a control is 5'-GCAAGCTGACCCGTGTTTCA-3' (SEQ ID NO: 13).
  • the nucleotide sequence of the cloned cDNA was confirmed using BigDye terminator kit version 3.1 (manufactured by Applied Biosystems) and ABI sequencer model 3130xl (manufactured by Applied Biosystems).
  • Lipofectamine 3000 (Thermo Fisher Scientific Co., Ltd.) was used as a vector, and the vector was introduced into cells according to the instruction manual.
  • BRQ Brekiner sodium salt
  • Example 1 Examination on cytotoxic activity of DHODH inhibitor against pluripotent stem cells
  • Mouse ES cells and mouse iPS cells were cultured for 3 days in the presence of DHODH inhibitors (BRQ, Leflunomide, Teriflunomide or Videfludimus), respectively.
  • the survival rate was examined by the MTT assay.
  • Example 2 Examination on cytotoxic activity of DHODH inhibitor on somatic stem cells, etc.
  • pluripotent stem cells NT2: human embryonal carcinoma (EC) cells
  • PA6 mouse bone marrow-derived stromal cells
  • C2C12 mouse myoblasts
  • Example 3 Verification of cell-specific damaging activity of DHODH inhibitor on pluripotent stem cells
  • Pluripotent stem cells mouse iPS cells
  • Somatic stem cells mouse neural stem cells
  • ES cells and iPS cells disappeared in the presence of 10 ⁇ M BRQ, but no apparent cytotoxicity to neural stem cells was observed. I could't do it.
  • Example 4 Verification of Nucleoside Neutralizing Activity Against Cytotoxicity of DHODH Inhibitors Pyrimidine synthesis is regulated by nascent (de novo synthesis) and salvage (salvage) pathways. DHODH is a key factor in de novo synthesis that catalyzes the fourth rate-limiting chemical reaction in the synthetic pathway. Therefore, it was investigated whether the effect of removing pluripotent stem cells by the DHODH inhibitor can be eliminated by adding a nucleoside such as uridine which is a starting material of the salvage pathway.
  • a nucleoside such as uridine which is a starting material of the salvage pathway.
  • nucleoside adenosine, guanosine, cytidine or uridine
  • uridine a nucleoside
  • FIGS. 7 and 8 it was revealed that pyrimidine nucleosides (uridine and cytidine) have a neutralizing activity, and particularly uridine has a strong neutralizing activity against the cytotoxic activity of BRQ.
  • UDP-GlcNac uridine diphosphate-N-acetylglucosamine
  • OHT O-linked N-acetylglucosaminyltransferase
  • DHODH inhibitors such as BRQ exert cytotoxic activity on pluripotent stem cells by inhibiting the pyrimidine synthesis pathway. It was also suggested that its cytotoxic activity could be suppressed by activating the salvage pathway.
  • Example 6 Verification of Effect of DHODH Inhibitor on Marker Molecule of Pluripotent Stem Cell
  • three transcription factors Sox2 involved in promotion of self-renewal ability and maintenance of undifferentiated state.
  • Oct4, Nanog are expressed at high levels. Therefore, the effect of the DHODH inhibitor on the marker molecules of these pluripotent stem cells was examined.
  • Sox2 in pluripotent stem cells mouse iPS cells and mouse ES cells
  • somatic stem cells mouse neural stem cells
  • pluripotent stem cells mouse iPS cells and mouse ES cells
  • BRQ pluripotent stem cell marker gene
  • Nanog which is normally localized in the nucleus of pluripotent stem cells, becomes spread and distributed in the cytoplasm by BRQ treatment.
  • Example 7 Nuclear export of CRM1-dependent pluripotent stem cell marker molecule induced by DHODH inhibitor Pluripotent stem cells in the presence or absence of BRQ or pan-caspase inhibitor Z-VAD Were cultured. On the second day after treatment with BRQ, the number of Oct4-positive cells and Nanog-positive cells in pluripotent stem cells (ES cells and iPS cells) was measured.
  • Oct4 positive cells in ES cells treated with BRQ 2%, Nanog positive cells: 6%.
  • Oct4 positive cells 0%, Nanog positive cells: 0% in iPS cells treated with BRQ.
  • DMSO Oct4-positive cells: 85%, Nanog-positive cells: 86% in the ES cells treated with, and Oct4-positive cells: 94%, Nanog-positive cells: 93% in the iPS cells treated with DMSO).
  • pluripotent stem cells were cultured in a medium supplemented with leptomycin B (LMB) in the presence or absence of BRQ, and nuclear localization of Nanog and Oct4 was analyzed.
  • LMB is a specific inhibitor of CRM1 also known as Exportin1.
  • Example 8 Verification of suppressive effect of DHODH inhibitor on tumorigenicity of pluripotent stem cells
  • the tumorigenicity of pluripotent stem cells pretreated with BRQ which is a DHODH inhibitor was examined. Specifically, first, after culturing in a medium supplemented with DMSO alone or 10 ⁇ M BRQ for 2 days, surviving pluripotent stem cells were subcutaneously injected into the buttocks of the NOD/SCID mice. Then, 4 weeks after the transplantation, the tumor was excised and observed.
  • pluripotent stem cells were first transplanted subcutaneously into the buttocks of NOD/SCID mice to form tumors reaching a size of more than 100 mm 3 . Then, BRQ was intraperitoneally injected once every three days, and a tumor was excised from the mouse on day 3 after the fifth intraperitoneal injection.
  • BRQ administration suppressed tumor growth. Although not shown in the figure, no visible side effect was observed in the BRQ-administered mice. As also shown in FIG. 28, teratomas size treated with DMSO and BRQ is each a 0.55 cm 3 and 0.12 cm 3 in those from ES cells, those derived from iPS cells, 1.37Cm 3 and It was 0.32 cm 3 .
  • Ki67-positive proliferating cells in BRQ-treated tumors were significantly reduced as compared with those in DMSO-treated tumors.
  • the number of Oct4-positive cells and Nanog-positive cells was significantly reduced in BRQ-treated tumors, but many cells expressing pluripotent stem cell markers were observed in DMSO-treated tumors. It was On the other hand, both tumors similarly contained cells expressing ATF, ⁇ III tubulin and SMA, which are markers in the three germ layers, respectively.
  • the DHODH inhibitor can suppress tumor formation by specifically removing undifferentiated pluripotent stem cells without causing obvious cytotoxicity to differentiated cells and mice.
  • the present invention it is possible to remove undifferentiated pluripotent stem cells without causing significant cytotoxicity to the differentiated cells. Therefore, the present invention is excellent in removing undifferentiated pluripotent stem cells remaining in a cell group differentiated from pluripotent stem cells, so that the risk of tumorigenesis is reduced, and regenerative medicine with few side effects, etc. Is useful in.

Abstract

The purpose of the present invention is to provide a composition and method for removing undifferentiated pluripotent stem cells that remain present in a cell group that has been induced to differentiate from pluripotent stem cells. It has been discovered that a dihydroorotate dehydrogenase inhibitor exhibits cytotoxicity for pluripotent stem cells while not exhibiting significant cytotoxicity for cells that have undergone differentiation, e.g., somatic stem cells.

Description

多能性幹細胞を除去するための組成物、及び多能性幹細胞の除去方法Composition for removing pluripotent stem cells and method for removing pluripotent stem cells
　本発明は、多能性幹細胞を除去するための組成物、及び多能性幹細胞の除去方法に関する。より詳しくは、多能性幹細胞から分化誘導した細胞群に残存する未分化の多能性幹細胞を除去するための組成物及び方法に関する。 The present invention relates to a composition for removing pluripotent stem cells and a method for removing pluripotent stem cells. More specifically, the present invention relates to a composition and method for removing undifferentiated pluripotent stem cells remaining in a cell group in which differentiation is induced from pluripotent stem cells.
　胚性幹細胞（ＥＳ細胞）、人工多能性幹細胞（ｉＰＳ細胞）等の多能性幹細胞は、生体を構成する全ての細胞に分化する能力を有することから再生医療実現化の要としてその利用が期待されている。そして、現在までに、これら多能性幹細胞から治療に必要とされる特定の移植用機能細胞への分化誘導法が多々確立されている。 Pluripotent stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) have the ability to differentiate into all the cells that make up the living body, and therefore they are used as the key to the realization of regenerative medicine. Is expected. In addition, until now, various methods for inducing differentiation from these pluripotent stem cells to specific functional cells for transplantation required for treatment have been established.
　しかしながら、分化誘導後に残存した未分化の多能性幹細胞は、腫瘍を形成する可能性がある。そのため、移植治療を進める上で大きな障害となっており、この問題を解決するため、多能性幹細胞を除去する様々な方法が開発されている（非特許文献１～５）。 However, undifferentiated pluripotent stem cells remaining after differentiation induction may form tumors. Therefore, it is a major obstacle to the promotion of transplantation therapy, and various methods for removing pluripotent stem cells have been developed to solve this problem (Non-Patent Documents 1 to 5).
　しかし、これらの方法による他の細胞への安全性についての検証は不十分なこともあり、副作用少なく、多能性幹細胞を除去する方法は未だ実用化されていないのが現状である。 However, the verification of the safety to other cells by these methods may be insufficient, there are few side effects, and the method of removing pluripotent stem cells has not yet been put to practical use.
　本発明は、前記従来技術の有する課題に鑑みてなされたものであり、未分化の多能性幹細胞に対して細胞傷害性をもたらす一方で、他の細胞に対しては毒性の少ない化合物を見出すことを目的とする。さらには、該化合物を有効成分とする多能性幹細胞を除去するための組成物、及び前記化合物を用いた多能性幹細胞の除去方法を提供することを、本発明の目的とする。 The present invention has been made in view of the above problems of the prior art, and finds a compound that brings cytotoxicity to undifferentiated pluripotent stem cells, while having little toxicity to other cells. The purpose is to Furthermore, it is an object of the present invention to provide a composition for removing pluripotent stem cells containing the compound as an active ingredient, and a method for removing pluripotent stem cells using the compound.
　本発明者は、前記目的を達成すべく鋭意研究を重ねた結果、ジヒドロオロト酸デヒドロゲナーゼ（ＤＨＯＤＨ）阻害剤が、ＥＳ細胞及びｉＰＳ細胞等の多能性幹細胞に対して細胞傷害活性を示すことを見出した。一方、ＤＨＯＤＨ阻害剤は、分化した細胞（アストロサイト等の体細胞、神経幹細胞等の体性幹細胞）に対しては、有意な細胞傷害性を示さないことを明らかにした。 The present inventors have conducted extensive studies to achieve the above-mentioned object, and as a result, found that a dihydroorotate dehydrogenase (DHODH) inhibitor shows cytotoxic activity against pluripotent stem cells such as ES cells and iPS cells. It was On the other hand, it was revealed that DHODH inhibitors do not show significant cytotoxicity to differentiated cells (somatic cells such as astrocytes and somatic stem cells such as neural stem cells).
　さらに、多能性幹細胞をＤＨＯＤＨ阻害剤にて処理した上でマウスに移植することによって、または多能性幹細胞を移植したマウスにＤＨＯＤＨ阻害剤を投与することによって、前記マウスに特段の副作用をもたらすことなく、多能性幹細胞からの腫瘍形成を抑制できることも確認した。 Furthermore, by treating pluripotent stem cells with a DHODH inhibitor and then transplanting them into a mouse, or by administering a DHODH inhibitor to a mouse into which a pluripotent stem cells are transplanted, a particular side effect is brought about in the mouse. It was also confirmed that the tumor formation from pluripotent stem cells could be suppressed without the above.
　ＤＨＯＤＨは、ピリミジンのｄｅ　ｎｏｖｏ合成の４番目の化学反応を触媒する酸化還元酵素である。ＤＨＯＤＨ阻害剤は、その合成を阻害することによって、Ｔ細胞やＢ細胞の増殖を抑制し、免疫抑制効果を発揮することが既に明らかとなっており、当該薬剤は、かかる効果故、関節リウマチ等の自己免疫疾患の治療に用いられている。さらに、ＤＨＯＤＨ阻害剤については、がん幹細胞における分化阻害を解除することによって、または同細胞におけるｐ５３の合成を増強することによって、白血病（急性骨髄性白血病、慢性骨髄性白血病）に対する治療効果を奏することも報告されている（非特許文献６及び７）。 DHODH is an oxidoreductase that catalyzes the fourth chemical reaction of de novo synthesis of pyrimidine. It has already been clarified that the DHODH inhibitor suppresses the proliferation of T cells and B cells and exerts an immunosuppressive effect by inhibiting the synthesis thereof, and the drug is due to such an effect, rheumatoid arthritis and the like. Is used for the treatment of autoimmune diseases of. Furthermore, DHODH inhibitors exert a therapeutic effect on leukemia (acute myeloid leukemia, chronic myelogenous leukemia) by releasing differentiation inhibition in cancer stem cells or by enhancing p53 synthesis in the same cells. It has also been reported (Non-patent documents 6 and 7).
　しかしながら、ＤＨＯＤＨ阻害剤の多能性幹細胞への影響については何ら検証も示唆もされていなかったものの、本発明者は、上述のとおり、当該薬剤によって多能性幹細胞を除去できることを初めて明らかにした。その一方で、多能性幹細胞と比して劣るとはいえ多分化能と自己増殖性を備え、同じく幹細胞に分類される、体性幹細胞（神経幹細胞等）に対しては、ＤＨＯＤＨ阻害剤は有意な細胞傷害性を示さないことも、本発明者は見出し、本発明を完成するに至った。 However, although no verification or suggestion was made on the effect of the DHODH inhibitor on pluripotent stem cells, the present inventor revealed for the first time that the drug can remove pluripotent stem cells, as described above. .. On the other hand, for somatic stem cells (neural stem cells, etc.), which are inferior to pluripotent stem cells but have pluripotency and self-proliferative ability and are also classified as stem cells, a DHODH inhibitor is The present inventors have found that they do not show significant cytotoxicity, and have completed the present invention.
　すなわち、本発明は、ＤＨＯＤＨ阻害剤を用いた、多能性幹細胞を除去するための組成物、及び多能性幹細胞の除去方法に関し、より具体的には以下を提供する。
＜１＞　ジヒドロオロト酸デヒドロゲナーゼ阻害剤を有効成分として含有する、多能性幹細胞から分化誘導した細胞群に残存する未分化の多能性幹細胞を除去するための組成物。
＜２＞　前記多能性幹細胞が、胚性幹細胞（ＥＳ細胞）、人工多能性幹細胞（ｉＰＳ細胞）及び胚性腫瘍細胞（ＥＣ細胞）からなる群から選択される少なくとも１の多能性幹細胞である、＜１＞に記載の組成物。
＜３＞　前記ジヒドロオロト酸デヒドロゲナーゼ阻害剤がブレキナー（ｂｒｅｑｕｉｎａｒ）である、＜１＞又は＜２＞に記載の組成物。
＜４＞　前記細胞群が、前記多能性幹細胞から分化誘導した体性幹細胞を含む細胞群である、＜１＞～＜３＞のうちのいずれか一項に記載の組成物。
＜５＞　多能性幹細胞から分化誘導した細胞群とジヒドロオロト酸デヒドロゲナーゼ阻害剤とを接触させる工程を含む、前記細胞群から残存する未分化の多能性幹細胞を除去する方法。
＜６＞　前記多能性幹細胞が、胚性幹細胞（ＥＳ細胞）、人工多能性幹細胞（ｉＰＳ細胞）及び胚性腫瘍細胞（ＥＣ細胞）からなる群から選択される少なくとも１の多能性幹細胞である、＜５＞に記載の方法。
＜７＞　前記ジヒドロオロト酸デヒドロゲナーゼ阻害剤がブレキナー（ｂｒｅｑｕｉｎａｒ）である、＜５＞又は＜６＞に記載の方法。
＜８＞　前記細胞群が、前記多能性幹細胞から分化誘導した体性幹細胞を含む細胞群である、＜５＞～＜７＞のうちのいずれか一項に記載の方法。 That is, the present invention relates to a composition for removing pluripotent stem cells and a method for removing pluripotent stem cells using a DHODH inhibitor, and more specifically provides the following.
<1> A composition for removing undifferentiated pluripotent stem cells remaining in a cell group differentiated from pluripotent stem cells, containing a dihydroorotic acid dehydrogenase inhibitor as an active ingredient.
<2> The pluripotent stem cell is at least one pluripotent stem cell selected from the group consisting of embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells) and embryonic tumor cells (EC cells) The composition according to <1>.
<3> The composition according to <1> or <2>, wherein the dihydroorotic acid dehydrogenase inhibitor is brequinar.
<4> The composition according to any one of <1> to <3>, wherein the cell group is a cell group containing somatic stem cells differentiated from the pluripotent stem cells.
<5> A method for removing residual undifferentiated pluripotent stem cells from the cell group, which comprises a step of contacting a cell group differentiated from the pluripotent stem cells with a dihydroorotate dehydrogenase inhibitor.
<6> The pluripotent stem cell is at least one pluripotent stem cell selected from the group consisting of embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells) and embryonic tumor cells (EC cells) The method according to <5>.
<7> The method according to <5> or <6>, wherein the dihydroorotic acid dehydrogenase inhibitor is brequinar.
<8> The method according to any one of <5> to <7>, wherein the cell group is a cell group containing somatic stem cells differentiated from the pluripotent stem cells.
　本発明によれば、分化細胞には有意な細胞傷害をもたらすことなく、未分化の多能性幹細胞を除去することが可能となる。すなわち、本発明によれば、多能性幹細胞から分化誘導した細胞群に残存する未分化の多能性幹細胞を除去することが可能となる。 According to the present invention, undifferentiated pluripotent stem cells can be removed without causing significant cytotoxicity to differentiated cells. That is, according to the present invention, it becomes possible to remove the undifferentiated pluripotent stem cells remaining in the cell group differentiated from the pluripotent stem cells.
ジヒドロオロト酸デヒドロゲナーゼ（ＤＨＯＤＨ）阻害剤存在下３日間培養した後の、マウス胚性幹（ＥＳ）細胞の生存率を示すグラフである。図中、「ＢＲＱ」、「Ｌｅｆｌｕｎｏｍｉｄｅ］、「Ｔｅｒｉｆｌｕｎｏｍｉｄｅ」及び「Ｖｉｄｏｆｌｕｄｉｍｕｓ」は、各ＤＨＯＤＨ阻害剤（ブレキナー、レフルノミド、テリフルノミド及びビドフルジムス）存在下における細胞の生存率を示す（図中の表記については、図２においても同様である）。1 is a graph showing the survival rate of mouse embryonic stem (ES) cells after culturing for 3 days in the presence of a dihydroorotic acid dehydrogenase (DHODH) inhibitor. In the figure, “BRQ”, “Leflunomide”, “Teriflunomide” and “Vidofludimus” indicate the viability of cells in the presence of each DHODH inhibitor (Brekiner, Leflunomide, Teriflunomide and Bidofludimus) (for the notation in the figure, The same applies to FIG. 2). ＤＨＯＤＨ阻害剤存在下３日間培養した後の、マウス人工多能性幹（ｉＰＳ）細胞の生存率を示すグラフである。It is a graph which shows the survival rate of mouse induced pluripotent stem (iPS) cells after 3 days of culture in the presence of a DHODH inhibitor. ＢＲＱ存在下３日間培養した後の、マウス神経幹細胞等の生存率を示すグラフである。図中、「ｍＮＳＣ」、「ｍＮＳＣ＋５％ＦＣＳ」及び「アストロサイト」は、ＢＲＱ存在下における、マウス神経幹細胞、５％ウシ胎仔血清添加時のマウス神経幹細胞及びマウスアストロサイトの生存率を各々示す。It is a graph which shows the survival rate of mouse neural stem cells etc. after 3 days of culture in the presence of BRQ. In the figure, “mNSC”, “mNSC+5% FCS”, and “astrocyte” represent the survival rates of mouse neural stem cells and mouse astrocytes in the presence of BRQ when 5% fetal bovine serum was added. １０μＭ　ＢＲＱ存在下３日間培養後の、各種細胞の生存率を示すグラフである。図中、「ＥＳ」、「ｉＰＳ」、「ＮＴ２」、「ＰＡ６」、「Ｃ２Ｃ１２」、「ＮＳＣ」及び「アストロサイト」は、マウスＥＳ細胞、マウスｉＰＳ細胞、ヒト胚性癌細胞、マウス骨髄由来ストローマ細胞、マウス筋芽細胞、マウス神経幹細胞及びマウスアストロサイトの前記生存率を各々示す。It is a graph showing the survival rate of various cells after 3 days of culture in the presence of 10 μM BRQ. In the figure, “ES”, “iPS”, “NT2”, “PA6”, “C2C12”, “NSC” and “astrosite” are derived from mouse ES cells, mouse iPS cells, human embryonal carcinoma cells and mouse bone marrow. The survival rates of stromal cells, mouse myoblasts, mouse neural stem cells and mouse astrocytes are shown respectively. ＢＲＱ存在下において、マウス神経幹細胞及びマウスＥＳ細胞を３日間混合培養した結果を示す、蛍光顕微鏡写真である。図中、「コントロール」はＢＲＱ非存在下における培養結果を示す。図中、上段の３写真は、神経幹細胞のマーカー（Ｎｅｓｔｉｎ）を免疫染色にて検出した結果を示す。中段の３写真は、多能性幹細胞のマーカー（Ｎａｎｏｇ）を免疫染色にて検出した結果を示す。下段の３写真は、Ｎｅｓｔｉｎ及びＮａｎｏｇを免疫染色にて検出した結果と、ＤＡＰＩによって細胞核を対比染色した結果とを重ね合わせた写真である。3 is a fluorescence micrograph showing the results of mixed culture of mouse neural stem cells and mouse ES cells for 3 days in the presence of BRQ. In the figure, "control" indicates the culture result in the absence of BRQ. In the figure, the upper three photographs show the results of detection of a neural stem cell marker (Nestin) by immunostaining. The middle three photographs show the results of detecting pluripotent stem cell markers (Nanog) by immunostaining. The bottom three photographs are photographs in which the results of detecting Nestin and Nanog by immunostaining and the results of counterstaining the cell nuclei with DAPI are superimposed. ＢＲＱ存在下において、マウス神経幹細胞及びマウスｉＰＳ細胞を３日間混合培養した結果を示す、蛍光顕微鏡写真である。図中、「コントロール」はＢＲＱ非存在下における培養結果を示す。図中、上段の３写真は、多能性幹細胞のマーカー（Ｎａｎｏｇ）を免疫染色にて検出した結果を示す。中段の３写真は、神経幹細胞のマーカー（Ｎｅｓｔｉｎ）を免疫染色にて検出した結果を示す。下段の３写真は、Ｎｅｓｔｉｎ及びＮａｎｏｇを免疫染色にて検出した結果と、ＤＡＰＩによって細胞核を対比染色した結果とを重ね合わせた写真である。3 is a fluorescence micrograph showing the results of mixed culture of mouse neural stem cells and mouse iPS cells for 3 days in the presence of BRQ. In the figure, "control" indicates the culture result in the absence of BRQ. In the figure, the upper three photographs show the results of detecting a pluripotent stem cell marker (Nanog) by immunostaining. The middle three photographs show the results of detecting a neural stem cell marker (Nestin) by immunostaining. The bottom three photographs are photographs in which the results of detecting Nestin and Nanog by immunostaining and the results of counterstaining the cell nuclei with DAPI are superimposed. １０μＭ　ＢＲＱ存在下、各種リボヌクレオシド（ウリジン、アデノシン、グアノシン又はシチジン）を添加した際の、マウスｉＰＳ細胞の生存率を示すグラフである。9 is a graph showing the survival rate of mouse iPS cells when various ribonucleosides (uridine, adenosine, guanosine or cytidine) were added in the presence of 10 μM BRQ. １０μＭ　ＢＲＱ存在下、各種リボヌクレオシド（ウリジン、アデノシン、グアノシン又はシチジン）を添加した際の、マウスＥＳ細胞の生存率を示すグラフである。6 is a graph showing the survival rate of mouse ES cells when various ribonucleosides (uridine, adenosine, guanosine or cytidine) were added in the presence of 10 μM BRQ. １０μＭ　ＢＲＱ存在下、各種リボヌクレオシド（ウリジン二リン酸（ＵＤＰ）又はウリジン二リン酸－Ｎ－アセチルグルコサミン（ＵＤＰ－ＧｌｃＮＡｃ））を添加した際の、マウスｉＰＳ細胞の生存率を示すグラフである。2 is a graph showing the survival rate of mouse iPS cells when various ribonucleosides (uridine diphosphate (UDP) or uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc)) were added in the presence of 10 μM BRQ. １０μＭ　ＢＲＱ存在下、各種リボヌクレオシド（ＵＤＰ又はＵＤＰ－ＧｌｃＮＡｃ）を添加した際の、マウスＥＳ細胞の生存率を示すグラフである。6 is a graph showing the survival rate of mouse ES cells when various ribonucleosides (UDP or UDP-GlcNAc) were added in the presence of 10 μM BRQ. 多能性幹細胞におけるＢＲＱ依存的細胞傷害性に対する、ヌクレオチド二リン酸の用量依存効果を示すグラフである。図中、「ＥＳＣ」はマウスＥＳ細胞に対する効果を示し、「ｉＰＳＣ」はマウスｉＰＳ細胞に対する効果を示す。また各グラフにおいて、丸はＵＤＰの、ダイアモンドはＣＤＰの、四角はＡＤＰの、三角はＧＤＰの用量依存効果を示す。エラーバーは±ＳＤ（標準偏差）を示す。統計学的有意性はｔ検定によって判定した。＊＊ｐ＜０．０１、＊＊＊ｐ＜０．００１。FIG. 6 is a graph showing the dose-dependent effect of nucleotide diphosphate on BRQ-dependent cytotoxicity in pluripotent stem cells. In the figure, “ESC” indicates the effect on mouse ES cells, and “iPSC” indicates the effect on mouse iPS cells. In each graph, the circles show the dose-dependent effects of UDP, the diamonds of CDP, the squares of ADP, and the triangles of GDP. Error bars indicate ±SD (standard deviation). Statistical significance was determined by t-test. ***P<0.01, ***p<0.001. ＢＲＱ存在下での、多能性幹細胞におけるＢｒｄＵ陽性細胞の割合を示すグラフである。図中、「ＥＳＣ」はマウスＥＳ細胞における前記割合を示し、「ｉＰＳＣ」はマウスｉＰＳ細胞細胞における前記割合を示す。「Ｎ」及び「ＢＲＱ」は、多能性幹細胞の培養培地においてＢＲＱが各々存在していない及び存在していることを示す。It is a graph which shows the ratio of BrdU positive cell in a pluripotent stem cell in presence of BRQ. In the figure, "ESC" indicates the ratio in mouse ES cells, and "iPSC" indicates the ratio in mouse iPS cell cells. "N" and "BRQ" indicate the absence and presence of BRQ in the culture medium of pluripotent stem cells, respectively. ＢＲＱ存在下での、多能性幹細胞におけるＣａｓｐ３陽性細胞の割合を示すグラフである。図中、「ＥＳＣ」はマウスＥＳ細胞における前記割合を示し、「ｉＰＳＣ」はマウスｉＰＳ細胞細胞における前記割合を示す。「Ｎ」及び「ＢＲＱ」は、多能性幹細胞の培養培地においてＢＲＱが各々存在していない及び存在していることを示す。It is a graph which shows the ratio of the Casp3 positive cell in a pluripotent stem cell in the presence of BRQ. In the figure, "ESC" indicates the ratio in mouse ES cells, and "iPSC" indicates the ratio in mouse iPS cell cells. "N" and "BRQ" indicate the absence and presence of BRQ in the culture medium of pluripotent stem cells, respectively. ＤＨＯＤＨ発現ベクターによるノックダウン効率をウエスタンブロッティングにより分析した結果を示す写真である。ＦＬＡＧタグ標識マウスＤＨＯＤＨ発現ベクターを、コントロールｓｈＲＮＡをコードするベクター（図中「Ｃ」）又はＤＨＯＤＨに対するｓｈＲＮＡ１～３を各々コードするベクター（図中、「ＤＨＯＤＨ　ｓｈ１，ｓｈ２、ｓｈ３」）と共にＣｏｓ７細胞に導入した。遺伝子導入してから２日後に、細胞抽出液を回収し、抗ＦＬＡＧ抗体及び抗ＧＡＰＤＨ抗体（ローディングコントロール）を用いたウエスタンブロッティングによって分析した。It is a photograph which shows the result of having analyzed the knockdown efficiency by a DHODH expression vector by western blotting. The FLAG-tagged mouse DHODH expression vector was transferred to a Cos7 cell together with a vector encoding a control shRNA (“C” in the figure) or a vector encoding shRNA1 to 3 for DHODH (“DHODH sh1, sh2, sh3” in the figure). Introduced. Two days after the gene transfer, the cell extract was collected and analyzed by Western blotting using anti-FLAG antibody and anti-GAPDH antibody (loading control). コントロールｓｈＲＮＡ発現多能性幹細胞又はＤＨＯＤＨｓｈＲＮＡ発現多能性幹細胞における、Ｋｉ６７陽性細胞の割合を示すグラフである。図中「ＥＳＣ」及び「ｉＰＳＣ」は、マウスＥＳ細胞及びマウスｉＰＳ細胞各々における前記割合を示す。エラーバーは±ＳＤを示す。統計学的有意性は、ｔ検定によって判定した。＊Ｐ＜０．０５，＊＊Ｐ＜０．０１，＊＊＊Ｐ＜０．００１。It is a graph which shows the ratio of Ki67 positive cell in a control shRNA expression pluripotent stem cell or a DHODHshRNA expression pluripotent stem cell. In the figure, "ESC" and "iPSC" indicate the above-mentioned proportions in mouse ES cells and mouse iPS cells, respectively. Error bars indicate ±SD. Statistical significance was determined by t-test. *P<0.05, **P<0.01, ***P<0.001. コントロールｓｈＲＮＡ発現多能性幹細胞又はＤＨＯＤＨｓｈＲＮＡ発現多能性幹細胞における、Ｃａｓｐ３陽性細胞の割合を示すグラフである。図中「ＥＳＣ」及び「ｉＰＳＣ」は、マウスＥＳ細胞及びマウスｉＰＳ細胞各々における前記割合を示す。エラーバーは±ＳＤを示す。統計学的有意性は、ｔ検定によって判定した。＊Ｐ＜０．０５，＊＊Ｐ＜０．０１，＊＊＊Ｐ＜０．００１。It is a graph which shows the ratio of a Casp3 positive cell in a control shRNA expression pluripotent stem cell or a DHODHshRNA expression pluripotent stem cell. In the figure, "ESC" and "iPSC" indicate the above-mentioned proportions in mouse ES cells and mouse iPS cells, respectively. Error bars indicate ±SD. Statistical significance was determined by t-test. *P<0.05, **P<0.01, ***P<0.001. コントロールｓｈＲＮＡ発現多能性幹細胞及びｄｈｏｄｈ　ｓｈ発現多能性幹細胞に、ＧＦＰ及びＫｉ６７を対象とする免疫染色を施して観察し、得られた代表的な結果を示す、蛍光顕微鏡写真である。図中「ＥＳＣ」及び「ｉＰＳＣ」は、マウスＥＳ細胞及びマウスｉＰＳ細胞各々を観察した結果を示す。１段目及び３段目における各３写真は、マウスＥＳ細胞及びマウスｉＰＳ細胞のＫｉ６７（赤色）の発現及び核（青色、ＤＡＰＩによる対比染色）を検出した結果を示す。２段目及び４段目における各３写真は、マウスＥＳ細胞及びマウスｉＰＳ細胞のＧＦＰ（緑色）及びＫｉ６７（赤色）の発現を検出した結果を示す。スケールバーは１００μｍを示す。Fig. 3 is a fluorescence micrograph showing representative results obtained by observing control shRNA-expressing pluripotent stem cells and dhodhsh-expressing pluripotent stem cells by immunostaining for GFP and Ki67. In the figure, “ESC” and “iPSC” indicate the results of observing mouse ES cells and mouse iPS cells, respectively. Each of the three photographs in the first and third rows shows the results of detecting Ki67 (red) expression and nuclei (blue, counterstaining with DAPI) in mouse ES cells and mouse iPS cells. Each of the three photographs in the second and fourth rows shows the results of detecting the expression of GFP (green) and Ki67 (red) in mouse ES cells and mouse iPS cells. The scale bar shows 100 μm. コントロールｓｈＲＮＡ発現多能性幹細胞及びｄｈｏｄｈ　ｓｈ発現多能性幹細胞に、ＧＦＰ及びＣａｓｐ３を対象とする免疫染色を施して観察し、得られた代表的な結果を示す、蛍光顕微鏡写真である。図中「ＥＳＣ」及び「ｉＰＳＣ」は、マウスＥＳ細胞及びマウスｉＰＳ細胞各々を観察した結果を示す。１段目及び３段目における各３写真は、マウスＥＳ細胞及びマウスｉＰＳ細胞のＣａｓｐ３（赤色）の発現及び核（青色、ＤＡＰＩによる対比染色）を検出した結果を示す。２段目及び４段目における各３写真は、マウスＥＳ細胞及びマウスｉＰＳ細胞のＧＦＰ（緑色）及びＣａｓｐ３（赤色）の発現を検出した結果を示す。スケールバーは１００μｍを示す。Fig. 3 is a fluorescence micrograph showing representative results obtained by observing control shRNA-expressing pluripotent stem cells and dhodhsh-expressing pluripotent stem cells by immunostaining for GFP and Casp3. In the figure, “ESC” and “iPSC” indicate the results of observing mouse ES cells and mouse iPS cells, respectively. The three photographs in the first and third rows show the results of detecting the expression of Casp3 (red) and the nucleus (blue, counterstaining with DAPI) of mouse ES cells and mouse iPS cells. The three photographs in the second and fourth rows show the results of detecting the expression of GFP (green) and Casp3 (red) in mouse ES cells and mouse iPS cells. The scale bar shows 100 μm. １０μＭ　ＢＲＱ存在下で２４時間培養した後に、各細胞を蛍光顕微鏡にて観察した結果を示す写真である。図中、「ＮＳＣ」、「ｉＰＳ」及び「ＥＳ」は、マウス神経幹細胞、マウスｉＰＳ細胞及びマウスＥＳ細胞を観察した結果を示す。「ＢＲＱ」は１０μＭ　ＢＲＱ存在下で培養した結果を示し、「ＤＭＳＯ」はＢＲＱ非存在下で培養した結果を示す。上段の６写真はＳｏｘ２を免疫染色した結果を示し、下段の６写真はＳｏｘ２を免疫染色した結果とＤＡＰＩによって細胞核を対比染色した結果とを重ね合わせた写真である。矢印はＳｏｘ２陰性細胞を示す。各数値はＤＡＰＩ陽性細胞に対するＳｏｘ２陽性細胞の割合（％）を示す。スケールバーは１００μｍを表す。After culturing for 24 hours in the presence of 10 μM BRQ, it is a photograph showing the results of observing each cell with a fluorescence microscope. In the figure, “NSC”, “iPS” and “ES” indicate the results of observing mouse neural stem cells, mouse iPS cells and mouse ES cells. “BRQ” indicates the result of culturing in the presence of 10 μM BRQ, and “DMSO” indicates the result of culturing in the absence of BRQ. The upper 6 photographs show the results of immunostaining Sox2, and the lower 6 photographs are the photographs in which the results of the immunostaining of Sox2 and the results of counterstaining the cell nuclei with DAPI are superimposed. Arrows indicate Sox2-negative cells. Each numerical value shows the ratio (%) of Sox2-positive cells to DAPI-positive cells. Scale bar represents 100 μm. １０μＭ　ＢＲＱ存在下で２４時間培養した後の各細胞における、Ｓｏｘ２陽性細胞の割合（％）を示すグラフである。図中、「ＮＳＣ」、「ｉＰＳ」及び「ＥＳ」は、マウス神経幹細胞、マウスｉＰＳ細胞及びマウスＥＳ細胞における各Ｓｏｘ２陽性細胞の割合を示す。「ＢＲＱ」は１０μＭ　ＢＲＱ存在下で培養した結果を示し、「Ｃｏｎｔ」はＢＲＱ非存在下で培養した結果を示す。「＊＊」はＰ値＜０．０１であることを示す。3 is a graph showing the ratio (%) of Sox2-positive cells in each cell after culturing for 24 hours in the presence of 10 μM BRQ. In the figure, “NSC”, “iPS” and “ES” indicate the proportion of each Sox2-positive cell in mouse neural stem cells, mouse iPS cells and mouse ES cells. “BRQ” indicates the result of culturing in the presence of 10 μM BRQ, and “Cont” indicates the result of culturing in the absence of BRQ. "**" indicates that P value <0.01. １０μＭ　ＢＲＱ存在下で１日又は２日培養した後の、各細胞における多能性幹細胞マーカー遺伝子（Ｓｏｘ２、Ｏｃｔ４及びＮａｎｏｇ）の発現量を示すグラフである。図中、「ＥＳ」及び「ｉＰＳ」は、マウスＥＳ細胞及びマウスｉＰＳ細胞における各遺伝子の発現量を示す。「ＢＲＱ」は１０μＭ　ＢＲＱ存在下で培養した結果を示し、「ＤＭＳＯ」はＢＲＱ非存在下で培養した結果を示す。各グラフの縦軸は、「ＤＭＳＯ」における各遺伝子の発現量を１００とした場合の相対的な値を示す。3 is a graph showing the expression levels of pluripotent stem cell marker genes (Sox2, Oct4 and Nanog) in each cell after culturing in the presence of 10 μM BRQ for 1 day or 2 days. In the figure, “ES” and “iPS” indicate the expression level of each gene in mouse ES cells and mouse iPS cells. “BRQ” indicates the result of culturing in the presence of 10 μM BRQ, and “DMSO” indicates the result of culturing in the absence of BRQ. The vertical axis of each graph shows a relative value when the expression level of each gene in "DMSO" is 100. １０μＭ　ＢＲＱ存在下で２日間培養した後に、各細胞を蛍光顕微鏡にて観察した結果を示す写真である。図中、「ＥＳ」及び「ｉＰＳ」は、マウスＥＳ細胞及びマウスｉＰＳ細胞を観察した結果を示す。「ＢＲＱ」は１０μＭ　ＢＲＱ存在下で培養した結果を示し、「ｃｏｎｔ」はＢＲＱ非存在下で培養した結果を示す。上段の４写真はＮａｎｏｇを免疫染色した結果を示し、下段の４写真はＮａｎｏｇを免疫染色した結果とＤＡＰＩによって細胞核を対比染色した結果とを重ね合わせた写真である。矢印はＮａｎｏｇが細胞質に広がって分布している細胞（Ｎａｎｏｇが核外に排除された細胞）を示す。After culturing in the presence of 10 μM BRQ for 2 days, it is a photograph showing the results of observing each cell with a fluorescence microscope. In the figure, "ES" and "iPS" indicate the results of observing mouse ES cells and mouse iPS cells. “BRQ” indicates the result of culturing in the presence of 10 μM BRQ, and “cont” indicates the result of culturing in the absence of BRQ. The upper four photographs show the results of immunostaining for Nanog, and the lower four photographs are the photographs in which the results of immunostaining of Nanog and the results of counterstaining cell nuclei with DAPI are superimposed. Arrows indicate cells in which Nanog is spread and distributed in the cytoplasm (cells in which Nanog is excluded from the nucleus). 多能性幹細胞（ＰＳＣ）を、ＤＭＳＯ、ＺＶＡＤ（１００ｎＭ）、ＢＲＱ（１０μＭ）又はＢＲＱ及びＺＶＡＤ（ＢＲＱ：１０μＭ、ＺＶＡＤ：１００ｎＭ）の存在下にて２日間培養し、Ｎａｎｏｇ及びＯｃｔ４を免疫標識した結果を示す、蛍光顕微鏡写真である。図中、「ＥＳＣ」及び「ｉＰＳＣ」は、マウスＥＳ細胞及びマウスｉＰＳ細胞を観察した結果を示す。図中の数値は、全細胞における各核ＰＳＣマーカー陽性細胞数の割合を示す。上段の８写真は、Ｏｃｔ４（緑色）を免疫染色した結果を示し、中段の８写真は、Ｎａｎｏｇ（赤色）を免疫染色した結果を示し、下段の８写真は、前記Ｏｃｔ４を免疫染色した結果及びＮａｎｏｇを免疫染色した結果に、ＤＡＰＩによって細胞核（青色）を対比染色した結果を重ね合わせた結果を示す。スケールバーは５０μｍを示す。Pluripotent stem cells (PSC) were cultured for 2 days in the presence of DMSO, ZVAD (100 nM), BRQ (10 μM) or BRQ and ZVAD (BRQ:10 μM, ZVAD:100 nM), and immunolabeled with Nanog and Oct4. It is a fluorescence micrograph which shows a result. In the figure, “ESC” and “iPSC” indicate the results of observing mouse ES cells and mouse iPS cells. The numerical values in the figure show the ratio of the number of cells positive for each nuclear PSC marker in all cells. The upper 8 photographs show the results of immunostaining Oct4 (green), the middle 8 photographs show the results of immunostaining Nanog (red), and the lower 8 photographs show the results of immunostaining Oct4 and The result of superimposing the result of counter-staining the cell nucleus (blue) with DAPI on the result of immunostaining Nanog is shown. The scale bar shows 50 μm. 多能性幹細胞（ＰＳＣ）を、ＤＭＳＯ、ＢＲＱ（１０μＭ）又はＢＲＱ及びＬＭＢ（ＢＲＱ：１０μＭ、ＬＭＢ：０．１５ｎＭ）の存在下にて２日間培養し、Ｎａｎｏｇ（赤色）及びＯｃｔ４（緑色）を免疫標識した結果を示す、蛍光顕微鏡写真である。図中、「ＥＳＣ」及び「ｉＰＳＣ」は、マウスＥＳ細胞及びマウスｉＰＳ細胞を各々観察した結果を示す。図中の数値は、全細胞における各核ＰＳＣマーカー陽性細胞数の割合を示す。上段の６写真は、Ｏｃｔ４（緑色）を免疫染色した結果を示し、中段の６写真は、Ｎａｎｏｇ（赤色）を免疫染色した結果を示し、下段の６写真は、前記Ｏｃｔ４を免疫染色した結果及びＮａｎｏｇを免疫染色した結果に、ＤＡＰＩによって細胞核（青色）を対比染色した結果を重ね合わせた結果を示す。スケールバーは１００μｍを示す。Pluripotent stem cells (PSC) were cultured in the presence of DMSO, BRQ (10 μM) or BRQ and LMB (BRQ:10 μM, LMB: 0.15 nM) for 2 days, and Nanog (red) and Oct4 (green) were cultured. It is a fluorescence micrograph which shows the result of immunolabeling. In the figure, "ESC" and "iPSC" indicate the results of observing mouse ES cells and mouse iPS cells, respectively. The numerical values in the figure show the ratio of the number of cells positive for each nuclear PSC marker in all cells. The upper 6 photographs show the results of immunostaining Oct4 (green), the middle 6 photographs show the results of immunostaining Nanog (red), and the lower 6 photographs show the results of immunostaining Oct4 and The results of immunostaining Nanog with the results of counterstaining the cell nuclei (blue) with DAPI are shown. The scale bar shows 100 μm. ＤＭＳＯ又はＢＲＱにより前処理した多能性幹細胞から形成されたテラトーマの代表的な写真を示す。図中、「ＥＳＣ」及び「ｉＰＳＣ」は、マウスＥＳ細胞及びマウスｉＰＳ細胞各々から形成されたテラトーマを観察した結果を示す。スケールバーは１ｃｍを示す。Representative photographs of teratomas formed from pluripotent stem cells pretreated with DMSO or BRQ are shown. In the figure, “ESC” and “iPSC” indicate the results of observing teratomas formed from mouse ES cells and mouse iPS cells, respectively. Scale bar indicates 1 cm. ＤＭＳＯ又はＢＲＱにより前処理した多能性幹細胞から形成された腫瘍の体積を示すグラフである。図中、「ＥＳＣ」及び「ｉＰＳＣ」は、マウスＥＳ細胞及びマウスｉＰＳ細胞各々から形成された腫瘍の体積を示す。エラーバーは±ＳＤを示す。統計学的有意性はｔ検定によって判定した。＊＊Ｐ＜０．０１，＊＊＊Ｐ＜０．００１。FIG. 6 is a graph showing the volume of tumors formed from pluripotent stem cells pretreated with DMSO or BRQ. In the figure, “ESC” and “iPSC” indicate the volume of tumor formed from mouse ES cells and mouse iPS cells, respectively. Error bars indicate ±SD. Statistical significance was determined by t-test. ***P<0.01, ***P<0.001. ＤＭＳＯ又はＢＲＱにより処理したマウスにおいて、多能性幹細胞から形成されたテラトーマの代表的な写真を示す。図中、「ＥＳＣ」及び「ｉＰＳＣ」は、マウスＥＳ細胞及びマウスｉＰＳ細胞各々から形成されたテラトーマを観察した結果を示す。スケールバーは１ｃｍを示す。Representative photographs of teratomas formed from pluripotent stem cells in mice treated with DMSO or BRQ are shown. In the figure, “ESC” and “iPSC” indicate the results of observing teratomas formed from mouse ES cells and mouse iPS cells, respectively. Scale bar indicates 1 cm. ＤＭＳＯ又はＢＲＱにより処理したマウスにおいて、多能性幹細胞から形成されたテラトーマの腫瘍体積を示すグラフである。図中、「ＥＳＣ」及び「ｉＰＳＣ」は、マウスＥＳ細胞及びマウスｉＰＳ細胞各々から形成された腫瘍体積を示す。エラーバーは±ＳＤを示す。統計学的有意性はｔ検定によって判定した。＊＊Ｐ＜０．０１，＊＊＊Ｐ＜０．００１。FIG. 6 is a graph showing the tumor volume of teratomas formed from pluripotent stem cells in mice treated with DMSO or BRQ. In the figure, “ESC” and “iPSC” indicate tumor volumes formed from mouse ES cells and mouse iPS cells, respectively. Error bars indicate ±SD. Statistical significance was determined by t-test. ***P<0.01, ***P<0.001. ＤＭＳＯ又はＢＲＱにより処理したマウスにおいて、多能性幹細胞から形成されたテラトーマにおけるＫｉ６７陽性細胞の割合示すグラフである。図中、「ＥＳＣ」及び「ｉＰＳＣ」は、マウスＥＳ細胞及びマウスｉＰＳ細胞各々から形成されたテラトーマにおける前記割合を示す。エラーバーは±ＳＤを示す。統計学的有意性はｔ検定によって判定した。＊＊Ｐ＜０．０１，＊＊＊Ｐ＜０．００１。6 is a graph showing the proportion of Ki67-positive cells in teratomas formed from pluripotent stem cells in mice treated with DMSO or BRQ. In the figure, "ESC" and "iPSC" indicate the above-mentioned proportions in teratoma formed from mouse ES cells and mouse iPS cells, respectively. Error bars indicate ±SD. Statistical significance was determined by t-test. ***P<0.01, ***P<0.001. Ｏｃｔ４（赤色）、Ｎａｎｏｇ（緑色）、ＡＦＰ（緑色）、βIIIチューブリン（赤色）及びＳＭＡ（緑色）を対象とする免疫染色を施したテラトーマについての代表的な画像を示す。図中、「ＥＳＣ」及び「ｉＰＳＣ」は、マウスＥＳ細胞及びマウスｉＰＳ細胞各々から形成されたテラトーマを観察した結果を示す。核はＤＡＰＩ（青色）にて対比染色によって検出した。スケールバーは１００μｍを示す。Representative images of teratoma immunostained for Oct4 (red), Nanog (green), AFP (green), βIII tubulin (red) and SMA (green) are shown. In the figure, “ESC” and “iPSC” indicate the results of observing teratomas formed from mouse ES cells and mouse iPS cells, respectively. Nuclei were detected by counterstaining with DAPI (blue). The scale bar shows 100 μm.
　後述の実施例に示すとおり、本発明者は、ジヒドロオロト酸デヒドロゲナーゼ（ＤＨＯＤＨ）の酵素活性を阻害し、ピリミジン合成を阻害することによって、多能性幹細胞に細胞傷害をもたらし、当該細胞を除去できることを明らかにした。また、その一方で、多能性幹細胞を分化誘導することによって得られえる体性幹細胞や体細胞等の分化細胞においては、ＤＨＯＤＨを阻害しても、有意な細胞傷害は生じないことも見出した。 As shown in Examples described below, the present inventors have shown that by inhibiting the enzymatic activity of dihydroorotate dehydrogenase (DHODH) and inhibiting pyrimidine synthesis, pluripotent stem cells can be cytotoxicized and removed. Revealed. On the other hand, it was also found that in somatic stem cells obtained by inducing differentiation of pluripotent stem cells or differentiated cells such as somatic cells, even if DHODH is inhibited, significant cytotoxicity does not occur. ..
　したがって、本発明は、ＤＨＯＤＨ阻害剤を用いる、多能性幹細胞から分化誘導した細胞群に残存する未分化の多能性幹細胞を除去するための組成物及び方法を、提供する。 Accordingly, the present invention provides a composition and a method for removing undifferentiated pluripotent stem cells remaining in a cell population differentiated from pluripotent stem cells using a DHODH inhibitor.
　本発明において、「ジヒドロオロト酸デヒドロゲナーゼ（ＤＨＯＤＨ）」は、ピリミジンのｄｅ　ｎｏｖｏ合成経路において第４反応であるジヒドロオロト酸からオロト酸への酸化を触媒する酵素を意味し、例えば、ジヒドロオロト酸デヒドロゲナーゼ（フマル酸）（ＥＣ番号：１．３．９８．１）、ジヒドロオロト酸デヒドロゲナーゼ（ＮＡＤ＋）（ＥＣ番号：１．３．１．１４）、ジヒドロオロト酸デヒドロゲナーゼ（ＮＡＤＰ＋）（ＥＣ番号：１．３．１．１５）、ジヒドロオロト酸デヒドロゲナーゼ（キノン）（ＥＣ番号：１．３．５．２）が挙げられる。 In the present invention, “dihydroorotic acid dehydrogenase (DHODH)” means an enzyme that catalyzes the oxidation of dihydroorotic acid to orotic acid, which is the fourth reaction in the de novo synthetic pathway of pyrimidine, and includes, for example, dihydroorotic acid dehydrogenase (fumaric acid). ) (EC number: 1.3.98.1), dihydroorotic acid dehydrogenase (NAD+) (EC number: 1.3.1.14), dihydroorotic acid dehydrogenase (NADP+) (EC number: 1.3.1.15). ), and dihydroorotic acid dehydrogenase (quinone) (EC number: 1.3.5.2).
　「ジヒドロオロト酸デヒドロゲナーゼ阻害剤〈ＤＨＯＤＨ阻害剤）」は、前記触媒反応を阻害する活性を有する化合物を意味する。なお、本発明における「阻害」には活性等の完全な阻害のみならず部分的な阻害（抑制）も含まれる。 “Dihydroorotic acid dehydrogenase inhibitor (DHODH inhibitor)” means a compound having an activity of inhibiting the catalytic reaction. The “inhibition” in the present invention includes not only complete inhibition of activity and the like but also partial inhibition (suppression).
　本発明にかかる「ＤＨＯＤＨ阻害剤」としては、後述の実施例に示すような、ブレキナー（ｂｒｅｑｕｉｎａｒ、ＢＲＱ、６－フルオロ－２－（２’－フルオロ－１，１’－ビフェニル－４－イル）－３－メチル－４－キノリン－カルボン酸ナトリウム塩）、レフルノミド（ｌｅｆｌｕｎｏｍｉｄｅ、５－メチル－Ｎ－［４－（トリフルオロメチル）フェニル］－イソオキサゾール－４－カルボキサミド）、テリフルノミド（ｔｅｒｉｆｌｕｎｏｍｉｄｅ、（２Ｚ）－２－シアノ－３－ヒドロキシ－Ｎ－［４－（トリフルオロメチル）フェニル］ブタ－２－エンアミド）、ビドフルジムス（ｖｉｄｏｆｌｕｄｉｍｕｓ、２－（３－フルオロ－３’－メトキシビフェニル－４－イルカルバモイル）－シクロペンタ－１－エンカルボン酸、ＡＳＬＡＮ００３（２－（３，５－ジフルオロ－３’メトキシビフェニル－４－イルアミノ）ニコチン酸）等が挙げられる。さらに、例えば、Ｊ　Ｍｅｄ　Ｃｈｅｍ．、２０１３　Ａｐｒ　２５；５６（８）：３１４８－６７に開示されている化合物１５～１１０、Ｅｕｒ　Ｊ　Ｍｅｄ　Ｃｈｅｍ．２０１２　Ｍａｒ；４９：１０２－９に開示されている化合物７ａ～７ｎ、Ｂｉｏｃｈｅｍｉｓｔｒｙ．２００８　Ａｕｇ　２６；４７（３４）：８９２９－３６に開示されている化合物　コード３～８、国際公開第２００８／０７７６３９号に開示されているアミノニコチン及びイソニコチン酸誘導体、Ｎｅｕｒｏ　Ｏｎｃｏｌ．２０１９　Ｓｅｐ　１０．ｐｉｉ：ｎｏｚ１７０．ｄｏｉ：１０．１０９３／ｎｅｕｏｎｃ／ｎｏｚ１７０．に開示されている化合物　１０５８０（後述のＩＣ５０：９ｎＭ）が挙げられる。 Examples of the “DHODH inhibitor” according to the present invention include brequinar (BRQ, 6-fluoro-2-(2′-fluoro-1,1′-biphenyl-4-yl), as shown in Examples below. -3-methyl-4-quinoline-carboxylic acid sodium salt), leflunomide (leflunomide, 5-methyl-N-[4-(trifluoromethyl)phenyl]-isoxazole-4-carboxamide), teriflunomide, (2Z )-2-Cyano-3-hydroxy-N-[4-(trifluoromethyl)phenyl]but-2-enamide), Vidofludimus, 2-(3-Fluoro-3′-methoxybiphenyl-4-ylcarbamoyl) )-Cyclopent-1-enecarboxylic acid, ASLAN003 (2-(3,5-difluoro-3′methoxybiphenyl-4-ylamino)nicotinic acid), etc. Further, for example, J Med Chem., 2013 Apr 25 56(8):3148-67, compounds 15-110, Eur J Med Chem. 2012 Mar; 49:102-9, compounds 7a-7n, Biochemistry. 2008 Aug 26;47(; 34): 8929-36, Codes 3-8, aminonicotine and isonicotinic acid derivatives disclosed in WO 2008/077639, Neuro Oncol. 2019 Sep 10. pii: noz170.doi: Compound 10580 (IC 50 : 9 nM described later) disclosed in 10.1093/neuonc/noz170.
　本発明にかかる「ＤＨＯＤＨ阻害剤」として、好ましくはヒト由来ＤＨＯＤＨに対する５０％阻害濃度（ＩＣ５０）が１００ｎＭ以下である化合物が好ましく、ＩＣ５０が５０ｎＭ以下である化合物がより好ましく、ＩＣ５０が２０ｎＭ以下である化合物がさらに好ましい。かかる化合物としては、例えば、以下の化合物が挙げられる（各化合物の構造及びヒト由来ＤＨＯＤＨに対するＩＣ５０を以下に示す）。 The “DHODH inhibitor” according to the present invention is preferably a compound having a 50% inhibitory concentration (IC 50 ) for human-derived DHODH of 100 nM or less, more preferably a compound having an IC 50 of 50 nM or less, and an IC 50 of 20 nM. The following compounds are more preferable. Examples of such compounds include the following compounds (the structure of each compound and the IC 50 for human-derived DHODH are shown below).
Figure JPOXMLDOC01-appb-C000001
Figure JPOXMLDOC01-appb-C000001
Figure JPOXMLDOC01-appb-C000002
Figure JPOXMLDOC01-appb-C000002
Figure JPOXMLDOC01-appb-C000003
Figure JPOXMLDOC01-appb-C000003
Figure JPOXMLDOC01-appb-C000004
Figure JPOXMLDOC01-appb-C000004
Figure JPOXMLDOC01-appb-C000005
Figure JPOXMLDOC01-appb-C000005
Figure JPOXMLDOC01-appb-C000006
Figure JPOXMLDOC01-appb-C000006
Figure JPOXMLDOC01-appb-C000007
Figure JPOXMLDOC01-appb-C000007
　ＤＨＯＤＨの酵素活性は、当業者であれば、例えば、ジクロロインドフェノール（ＤＣＩＰ）を用いる色素還元アッセイにより求めることができる。吸収極大を６００ｎｍに持つＤＣＩＰは、ＤＨＯＤＨによる基質の酸化及び電子受容体の還元に伴い、還元され無色となる。そのため、当該波長における吸光度の減少を指標として、ＤＨＯＤＨの活性を求めることができる。また、ＤＨＯＤＨに対するＩＣ５０は、例えば、ヒト由来のＤＨＯＤＨ、基質（ジヒドロオロト酸）、電子受容体（例えば、ＣｏＱ）、及び緩衝液を含む酵素反応液に、様々な濃度の供試化合物（ＤＨＯＤＨ阻害剤）を添加し、前記ＤＣＩＰアッセイを行なうことにより求めることができる（国際公開第２００８／０７７６３９号、Ｂｉｏｃｈｅｍ　Ｊ．１９９８　Ｄｅｃ　１；３３６（Ｐｔ２）：２９９－３０３　参照のほど）。 The enzyme activity of DHODH can be determined by those skilled in the art by, for example, a dye reduction assay using dichloroindophenol (DCIP). DCIP having an absorption maximum at 600 nm is reduced and becomes colorless due to the oxidation of the substrate by DHODH and the reduction of the electron acceptor. Therefore, the activity of DHODH can be determined using the decrease in the absorbance at the wavelength as an index. In addition, the IC 50 for DHODH is, for example, that DOHDH derived from human, a substrate (dihydroorotic acid), an electron acceptor (for example, CoQ), and an enzyme reaction solution containing a buffer have various concentrations of a test compound (DHODH inhibition). Agent) and perform the DCIP assay (see WO 2008/077639, Biochem J. 1998 Dec 1;336(Pt2):299-303).
　また、本発明にかかる「ＤＨＯＤＨ阻害剤」には、その阻害活性を有する限り、薬理学上許容される塩、水和物又は溶媒和物も含まれる。このような薬理学上許容される塩としては、特に制限はなく、当該薬剤の各構造等に応じて適宜選択することができ、例えば、酸付加塩（塩酸塩、硫酸塩、臭化水素塩、硝酸塩、硫酸水素酸塩、リン酸塩、酢酸塩等）、塩基付加塩（ナトリウム塩、カリウム塩、亜鉛塩、カルシウム塩等）が挙げられる。また、水和物又は溶媒和物としては、特に制限はなく、例えば、ＤＨＯＤＨ阻害剤又はその塩１分子に対し、０．１～１０分子の水又は溶媒が付加したものが挙げられる。 The “DHODH inhibitor” according to the present invention also includes a pharmacologically acceptable salt, hydrate or solvate as long as it has the inhibitory activity. Such a pharmacologically acceptable salt is not particularly limited and can be appropriately selected according to each structure of the drug, and examples thereof include acid addition salts (hydrochloride, sulfate, hydrobromide). , Nitrates, hydrogensulfates, phosphates, acetates, etc.) and base addition salts (sodium salts, potassium salts, zinc salts, calcium salts, etc.). The hydrate or solvate is not particularly limited, and examples thereof include those in which 0.1 to 10 molecules of water or solvent are added to 1 molecule of DHODH inhibitor or its salt.
　さらに、本発明にかかる「ＤＨＯＤＨ阻害剤」には、その阻害活性を有する限り、互変異性体、幾何異性体、不斉炭素に基づく光学異性体、立体異性体等の総ての異性体及び異性体混合物が含まれる。さらにまた、ＤＨＯＤＨ阻害剤が生体内で酸化、還元、加水分解、アミノ化、脱アミノ化、水酸化、リン酸化、脱水酸化、アルキル化、脱アルキル化、抱合等の代謝を受けてなおその阻害活性を示す化合物をも包含し、また本発明は生体内で酸化、還元、加水分解等の代謝を受けてＤＨＯＤＨ阻害剤を生成する化合物をも包含する。 Furthermore, the “DHODH inhibitor” according to the present invention includes all isomers such as tautomers, geometric isomers, optical isomers based on asymmetric carbons, stereoisomers and the like, as long as it has the inhibitory activity. Includes isomer mixtures. Furthermore, DHODH inhibitors undergo metabolism in vivo such as oxidation, reduction, hydrolysis, amination, deamination, hydroxylation, phosphorylation, dehydroxylation, alkylation, dealkylation, conjugation, etc., and still inhibit the metabolism. The present invention also includes compounds that exhibit activity, and the present invention also includes compounds that are metabolized in vivo by oxidation, reduction, hydrolysis and the like to produce DHODH inhibitors.
　なお、ＤＨＯＤＨ阻害剤に関しては、上述のとおり、多くの化合物が開発され市販もされているため、購入することによって入手することができる。また市販されていなくとも、それら化合物の製造方法についても多々報告がなされている。そのため、当業者であれば、当該製造方法に沿って適宜調製することもできる。 Regarding the DHODH inhibitor, many compounds have been developed and are on the market as described above, so they can be obtained by purchasing. Many reports have been made on methods for producing these compounds even if they are not commercially available. Therefore, a person skilled in the art can also appropriately prepare according to the manufacturing method.
　本発明において「多能性幹細胞」とは、分化万能性及び自己複製能を備えている細胞であればよく、例えば、胚性幹細胞（ＥＳ細胞）、胚性腫瘍細胞（ＥＣ細胞）、エピブラスト幹細胞（ＥｐｉＳ細胞）、胚性生殖細胞（ＥＧ細胞）、多能性生殖細胞（ｍＧＳ細胞）、ＭＵＳＥ細胞（Ｋｕｒｏｄａ　Ｙ．ら、Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．Ｕ．Ｓ．Ａ．、２０１０年、１０７巻、１９号、８６３９～８６４３ページ　参照）等の生体から採取され得る細胞が挙げられる。さらに、「多能性幹細胞」には、人工多能性幹細胞（ｉＰＳ細胞）のように、生体から採取された体細胞から人工的に分化多能性等を持たせるよう誘導された細胞も含まれる。また、これら細胞の由来としては特に制限はなく、ヒト及び非ヒト動物（例えば、マウス及びラット等のげっ歯類、ウシ、ウマ、ブタ、ヒツジ、サル、イヌ、並びにネコ等の哺乳類、ニワトリ等の鳥類）が挙げられる。さらに、後述の再生医療等を目的とする場合には、免疫拒絶反応を抑えるという観点から後述の分化細胞を移植する対象に由来する多能性幹細胞であることが望ましい。 In the present invention, the “pluripotent stem cell” may be any cell having pluripotency and self-renewal ability, and examples thereof include embryonic stem cells (ES cells), embryonal tumor cells (EC cells), and epiblasts. Stem cells (EpiS cells), embryonic germ cells (EG cells), pluripotent germ cells (mGS cells), MUSE cells (Kuroda Y. et al., Proc. Natl. Acad. Sci. USA, 2010) , Vol. 107, No. 19, pp. 8639-8643). Furthermore, "pluripotent stem cells" also include cells derived from somatic cells collected from a living body so as to have artificial pluripotency, such as induced pluripotent stem cells (iPS cells). Be done. The origin of these cells is not particularly limited, and humans and non-human animals (for example, rodents such as mice and rats, cows, horses, pigs, sheep, monkeys, dogs, mammals such as cats, chickens, etc.) Birds). Furthermore, in the case of aiming at the regenerative medicine described below, pluripotent stem cells derived from the target to which the differentiated cells described below are transplanted are desirable from the viewpoint of suppressing immune rejection.
　本発明において「未分化の多能性幹細胞」としては、分化しておらず、分化万能性を維持している前述の多能性幹細胞の他、多能性幹細胞が腫瘍化した細胞（例えば、テラトーマ、テトラカルシノーマ）も含まれる。 In the present invention, "undifferentiated pluripotent stem cells" include, in addition to the pluripotent stem cells that have not been differentiated and maintain pluripotency, cells in which pluripotent stem cells have turned into tumors (for example, Teratoma, tetracarcinoma) are also included.
　本発明において「多能性幹細胞から分化誘導した細胞群」とは、上述の多能性幹細胞に由来する任意の分化細胞を含む細胞の集団を意味する。かかる細胞群は、単種の分化細胞を含むものであってもよく、複数種の分化細胞を含むものであってもよい。さらに、その形態としては、それら細胞によって構成される組織、器官であってもよい。 In the present invention, the “cell group differentiated from pluripotent stem cells” means a population of cells including any differentiated cells derived from the above pluripotent stem cells. Such a cell group may include a single type of differentiated cell or may include a plurality of types of differentiated cells. Further, the form may be a tissue or an organ composed of these cells.
　本発明において「分化細胞」には、多能性を完全に喪失した細胞（最終分化細胞、例えば、体細胞、生殖細胞）のみならず、多能性を部分的に喪失した細胞（例えば、前駆細胞、体性幹細胞）も含まれる。「体性幹細胞」は、成体幹細胞、組織幹細胞とも称される細胞であり、例えば、神経幹細胞、衛星細胞、造血幹細胞（骨髄幹細胞）、間葉系幹細胞、腸管幹細胞、毛包幹細胞、乳腺幹細胞、内皮幹細胞、嗅粘膜幹細胞、神経冠幹細胞、精巣細胞が挙げられる。 In the present invention, "differentiated cells" include not only cells that have completely lost pluripotency (terminally differentiated cells, such as somatic cells and germ cells) but also cells that have partially lost pluripotency (eg, progenitor cells). Cells, somatic stem cells) are also included. "Somatic stem cells" are cells also called adult stem cells and tissue stem cells, and include, for example, neural stem cells, satellite cells, hematopoietic stem cells (bone marrow stem cells), mesenchymal stem cells, intestinal stem cells, hair follicle stem cells, mammary stem cells, Examples include endothelial stem cells, olfactory mucosa stem cells, neural crest stem cells, and testis cells.
　「分化細胞」への多能性幹細胞からの分化誘導は、通常、各分化細胞が発生する胚葉（外胚葉、中胚葉、内胚葉）への分化誘導を介して行なわれ、当業者であれば、各胚葉系細胞への分化誘導に適した低分子化合物、タンパク質等（分化誘導因子）を用いた公知の方法を、適宜選択して行なうことができる。例えば、神経細胞等の外胚葉系細胞へは、ＢＭＰ阻害剤、ＴＧＦβ及びアクチビン阻害剤の存在下にて培養することによって分化誘導することができる（Ｎａｔ　Ｂｉｏｔｅｃｈｎｏｌ．２００９　Ｍａｒ；２７（３）：２７５－８０　参照）。軟骨細胞等の中胚葉系の細胞へは、例えば、多能性幹細胞を、Ｗｎｔ及びアクチビンの存在下にて培養した後、ＢＭＰ４の存在下にて培養することにより、分化誘導することができる（Ｄｅｖｅｌｏｐｍｅｎｔ．２００８　Ｓｅｐ；１３５（１７）：２９６９－７９　参照）。また例えば、膵臓細胞、腸細胞等の内胚葉系細胞は、多能性幹細胞をアクチビンの存在下にて培養した後、ＢＭＰ４の存在下にて培養することによって、分化誘導することができる（例えば、Ｄｉａｂｅｔｅｓ　２０１０　Ｓｅｐ；５９（９）：２０９４－２１０１、Ｎａｔｕｒｅ．　２０１１　Ｆｅｂ　３；４７０（７３３２）：１０５－９　参照）。 Induction of differentiation of pluripotent stem cells into "differentiated cells" is usually carried out through induction of differentiation into germ layers (ectodermal, mesoderm, endoderm) in which each differentiated cell develops. A known method using a low-molecular compound, a protein or the like (differentiation inducing factor) suitable for inducing differentiation into each germ layer cell can be appropriately selected and performed. For example, ectodermal cells such as nerve cells can be induced to differentiate by culturing in the presence of a BMP inhibitor, TGFβ and an activin inhibitor (Nat Biotechnol. 2009 Mar; 27(3):275. -80). Differentiation can be induced into mesodermal cells such as chondrocytes by culturing pluripotent stem cells in the presence of Wnt and activin, and then culturing them in the presence of BMP4 ( Development.2008 Sep;135(17):2969-79). Further, for example, endodermal cells such as pancreatic cells and intestinal cells can be induced to differentiate by culturing pluripotent stem cells in the presence of activin and then culturing them in the presence of BMP4 (for example, , Diabetes 2010 Sep; 59(9):2094-2101, Nature. 2011 Feb 3; 470(7332): 105-9).
　また、本発明において、上述の細胞群に残存する未分化の多能性幹細胞の「除去」は、当該細胞の完全な除去のみならず、当該細胞の数又は前記細胞群における割合の低減を意味する。また、当該除去は、ｉｎ　ｖｉｔｒｏで行なわれるものであってもよく、ｉｎ　ｖｉｖｏ又はｅｘ　ｖｉｖｏで行なわれるものであってもよい。 Further, in the present invention, “removal” of undifferentiated pluripotent stem cells remaining in the above-mentioned cell group means not only complete removal of the cells but also reduction of the number of the cells or the ratio in the cell group. To do. In addition, the removal may be performed in vitro, or may be performed in vivo or ex vivo.
　（多能性幹細胞を除去するための組成物）
　本発明の実施形態として、ＤＨＯＤＨ阻害剤を有効成分として含有する、多能性幹細胞から分化誘導した細胞群に残存する未分化の多能性幹細胞を除去するための組成物が挙げられる。 (Composition for removing pluripotent stem cells)
Embodiments of the present invention include a composition containing a DHODH inhibitor as an active ingredient for removing undifferentiated pluripotent stem cells remaining in a cell group differentiated from pluripotent stem cells.
　本発明の組成物は、上述のＤＨＯＤＨ阻害剤の他、生理学的に許容される担体を含むものであってもよい。生理学的に許容される担体としては、例えば、生理的な等張液（生理食塩水、培地、ブドウ糖やその他の補助薬（Ｄ－ソルビトール、Ｄ－マンニトール、塩化ナトリウム等）を含む等張液等）、賦形剤、防腐剤、安定剤（ヒト血清アルブミン、ポリエチレングリコール等）、結合剤、溶解補助剤、非イオン性界面活性剤、緩衝剤（リン酸塩緩衝液、酢酸ナトリウム緩衝液等）、保存剤、酸化防止剤が挙げられる。 The composition of the present invention may contain a physiologically acceptable carrier in addition to the above-mentioned DHODH inhibitor. Examples of physiologically acceptable carriers include physiological isotonic solutions (physiological saline, culture medium, glucose and other isotonic solutions containing D-sorbitol, D-mannitol, sodium chloride, etc.). ), excipients, preservatives, stabilizers (human serum albumin, polyethylene glycol, etc.), binders, solubilizing agents, nonionic surfactants, buffers (phosphate buffer, sodium acetate buffer, etc.) , Preservatives and antioxidants.
　本発明の組成物は、研究目的又は医療目的（例えば、分化細胞、特に腫瘍化リスクの低減された分化細胞の製造）に用いられる試薬の形態であり得る。また、後述のとおり、前記分化細胞を移植した対象に投与される、医薬組成物の形態でもあり得る。 The composition of the present invention may be in the form of a reagent used for research purposes or medical purposes (for example, production of differentiated cells, particularly differentiated cells with reduced tumorigenicity risk). Further, as described below, it may be in the form of a pharmaceutical composition to be administered to a subject into which the differentiated cells have been transplanted.
　また、本発明は、分化細胞、特に腫瘍化リスクの低減された分化細胞を製造するためのキットをも提供する。当該キットにおいては、前記組成物の他、多能性幹細胞、上述の分化誘導因子、分化誘導用培地、培養器等の、多能性幹細胞から所望の細胞への分化誘導に必要な材料を含めることができる。さらに、所望の細胞に分化誘導できたことを確認するための、当該細胞特異的マーカー分子を検出するための試薬（例えば、当該マーカー分子に対して特異的な抗体）も、本発明のキットに含めることもできる。また、本発明の組成物の使用方法、分化誘導方法等を示した説明書も本発明のキットに含まれる。 The present invention also provides a kit for producing a differentiated cell, particularly a differentiated cell with reduced tumorigenicity risk. In the kit, in addition to the composition, pluripotent stem cells, the above-mentioned differentiation-inducing factor, differentiation-inducing medium, incubator, and other materials necessary for inducing differentiation of pluripotent stem cells into desired cells are included. be able to. Furthermore, a reagent for detecting the cell-specific marker molecule (for example, an antibody specific to the marker molecule) for confirming that differentiation into desired cells can be induced is also included in the kit of the present invention. It can also be included. In addition, the kit of the present invention also includes instructions indicating the method of using the composition of the present invention, the method of inducing differentiation and the like.
　（多能性幹細胞を除去するための方法）
　本発明の実施形態としてはまた、多能性幹細胞から分化誘導した細胞群とジヒドロオロト酸デヒドロゲナーゼ阻害剤とを接触させる工程を含む、前記細胞群から残存する未分化の多能性幹細胞を除去する方法が挙げられる。 (Method for removing pluripotent stem cells)
The embodiment of the present invention also includes a step of contacting a group of cells differentiated from pluripotent stem cells with a dihydroorotic acid dehydrogenase inhibitor, a method of removing undifferentiated pluripotent stem cells remaining from the group of cells Are listed.
　上述のＤＨＯＤＨ阻害剤と細胞群との「接触」については特に制限はなく、例えば、当該細胞群を維持するための培地にＤＨＯＤＨ阻害剤を添加することによって行なうことができる。その際の添加濃度としては、特に制限はなく、当業者であれば、対象とする多能性幹細胞及び細胞群の種類、並びに用いるＤＨＯＤＨ阻害剤の種類等によって適宜調整することができるが、通常０．１～１０００μＭであり、好ましくは１～１００μＭ、さらに好ましくは１０～５０μＭである。 There is no particular limitation on the “contact” between the above-mentioned DHODH inhibitor and the cell group, and for example, it can be performed by adding the DHODH inhibitor to the medium for maintaining the cell group. The concentration to be added at that time is not particularly limited and can be appropriately adjusted by those skilled in the art depending on the types of the pluripotent stem cells and cell groups of interest, the type of DHODH inhibitor to be used, etc. It is 0.1 to 1000 μM, preferably 1 to 100 μM, and more preferably 10 to 50 μM.
　なお、このように未分化の多能性幹細胞を除去することにより、再生医療等に有用な、腫瘍化リスクの低減された分化細胞を得ることができる。したがって、本発明の方法は、「多能性幹細胞から分化誘導した細胞群とジヒドロオロト酸デヒドロゲナーゼ阻害剤とを接触させる工程を含む、腫瘍化リスクの低減された分化細胞を製造する方法」とも言い換えることができる。 By removing undifferentiated pluripotent stem cells in this manner, differentiated cells with a reduced risk of tumorigenesis, which are useful for regenerative medicine and the like, can be obtained. Therefore, the method of the present invention can be rephrased as "a method for producing differentiated cells with reduced tumorigenicity risk, which comprises the step of contacting a cell group differentiated from pluripotent stem cells with a dihydroorotate dehydrogenase inhibitor" You can
　また、再生医療等のため、前記分化細胞を対象（ヒト又は非ヒト動物）に移植した場合には、ＤＨＯＤＨ阻害剤を当該対象に投与することによって、上述のＤＨＯＤＨ阻害剤と細胞群との「接触」を行なうことができる。投与形態としては特に制限はなく、静脈内投与、動脈内投与、腹腔内投与、皮下投与、皮内投与、気道内投与、直腸投与及び筋肉内投与、輸液による投与、局所投与、経口投与が挙げられ、移植する細胞群の種類、移植部位等に応じて適宜選択される。また、投与量は、対象の種類、年齢、体重、症状及び健康状態、投与形態、対象とする多能性幹細胞及び細胞群の種類、並びに用いるＤＨＯＤＨ阻害剤の種類等によって適宜調整されるが、１回あたりの投与量は、通常、０．００１～１０００ｍｇ／ｋｇ体重であり、好ましくは、０．１～１００ｍｇ／ｋｇ体重であり、より好ましくは、１～５０ｍｇ／ｋｇ体重である。また、１日あたりの投与回数としても、特に制限はなく、前記のような様々な要因を考慮して、適宜調整される。さらに、対象への投与は継続して行なわれてもよい（例えば、１週間毎に１回投与）。 Further, when the differentiated cells are transplanted into a subject (human or non-human animal) for regenerative medicine or the like, the DHODH inhibitor and the cell group can be treated by administering the DHODH inhibitor to the subject. Contact can be made. The dosage form is not particularly limited, and examples thereof include intravenous administration, intraarterial administration, intraperitoneal administration, subcutaneous administration, intradermal administration, respiratory tract administration, rectal administration and intramuscular administration, administration by infusion, local administration, and oral administration. It is appropriately selected depending on the type of cell group to be transplanted, the site of transplantation and the like. The dose is appropriately adjusted depending on the type of subject, age, weight, symptoms and health condition, administration form, types of target pluripotent stem cells and cell groups, types of DHODH inhibitors used, and the like. The dose per administration is usually 0.001 to 1000 mg/kg body weight, preferably 0.1 to 100 mg/kg body weight, and more preferably 1 to 50 mg/kg body weight. The number of administrations per day is also not particularly limited, and may be adjusted appropriately in consideration of various factors as described above. Further, the subject may be continuously administered (eg, once a week).
　また、未分化の多能性幹細胞を除去するための、ＤＨＯＤＨ阻害剤と細胞群との接触時間としては、当該除去が十分に行なわれる時間であればよく、当業者であれば適宜調整することができるが、通常２～７日間であり、好ましくは２～５日間であり、より好ましくは２～３日間である。 Further, the contact time between the DHODH inhibitor and the cell population for removing the undifferentiated pluripotent stem cells may be any time sufficient for the removal, and those skilled in the art can appropriately adjust the time. However, it is usually 2 to 7 days, preferably 2 to 5 days, and more preferably 2 to 3 days.
　以下、実施例に基づいて本発明をより具体的に説明するが、本発明は以下の実施例に限定されるものではない。また、本実施例は、以下の材料及び方法を用いて行なった。 Hereinafter, the present invention will be described more specifically based on examples, but the present invention is not limited to the following examples. In addition, this example was performed using the following materials and methods.
　（動物・化学試薬）
　マウスを用いた全ての実験は、北海道大学動物実験委員会が承認したプロトコールに従って行った。マウスは株式会社ホクドーから購入したものを用いた。特に断りがない限り、化学薬品及び増殖因子は、各々Ｉｎｖｉｔｒｏｇｅｎ社及びＰｅｐｒｏＴｅｃｈ社から購入したものを用いた。 (Animal/Chemical reagents)
All experiments using mice were performed according to the protocol approved by the Hokkaido University Animal Experiment Committee. The mouse used was purchased from Hokudo Co., Ltd. Unless otherwise specified, chemicals and growth factors used were those purchased from Invitrogen and PeproTech, respectively.
　（多能性幹細胞）
　マウスＥＳ細胞は、升井伸治氏（当該細胞を譲り受け時、京都大学ｉＰＳ研究所に在籍）より譲り受けた細胞株（Ｅ１４ｔｇ２ａ）を用いた。マウスｉＰＳ細胞は、理化学研究所バイオリソース研究センターから購入した細胞株（ｉＰＳ－Ｈｅｐ－ＦＢ／Ｎｇ／ｇｆｐ－１０３ｃ－１）を用いた。 (Pluripotent stem cells)
As the mouse ES cells, a cell line (E14tg2a) given by Mr. Shinji Masui (at the time of receiving the cells, enrolled in Kyoto University iPS Laboratory) was used. As the mouse iPS cells, a cell line (iPS-Hep-FB/Ng/gfp-103c-1) purchased from RIKEN BioResource Research Center was used.
　これら多能性幹細胞は、ＳＩＧＭＡ社のウェブサイトで公開している方法にて培養した（ｈｔｔｐｓ：／／ｗｗｗ．ｓｉｇｍａａｌｄｒｉｃｈ．ｃｏｍ／ｌｉｆｅ－ｓｃｉｅｎｃｅ／ｓｔｅｍ－ｃｅｌｌ－ｂｉｏｌｏｇｙ／ｓｔｅｍ－ｃｅｌｌ－ｐｒｏｔｏｃｏｌｓ．ｈｔｍｌ　参照）。また培養には、以下の組成からなる培養液（ＥＳＣ培養液）を用いた。
１ｍＭ　ピルビン酸ナトリウム（Ｇｉｂｃｏ社製，カタログ番号：１１３６０－０７０）、１ｘ非必須アミノ酸（Ｇｉｂｃｏ社製，カタログ番号１１１４０－０５０）、１００μＭ　２－メルカプトエタノール（ＳＩＧＭＡ社製，ＣＡＳ番号：６０－２４－２）、１０００ｕｎｉｔｓ／ｍＬ　白血病阻害因子（ＬＩＦ、ＥＳＧＲＯ（登録商標），Ｇｉｂｃｏ社製，カタログ番号：ＥＳＧ１１０７）、１５％　ノックアウト血清代替物（ＫＳＲ、Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社製，カタログ番号：１０８２８０２８）、及び１％　ウシ胎仔血清（ＦＢＳ、Ｈｙｃｌｏｎｅ社製）を含む、グラスゴー最小必須培地（ＧＭＥＭ培地、Ｓｉｇｍａ社製，製品番号：Ｇ５１５４－５００ＭＬ）。さらに、これら細胞を維持培養するために、１００ｍｍ培養ディッシュ（Ｆａｌｃｏｎ社製）の表面を、ウシ表皮由来のゼラチン（Ｓｉｇｍａ社製，製品番号：Ｇ９３９１）にてコートし、３７℃にて２時間以上置いたものを用いた。 These pluripotent stem cells were cultured by the method disclosed on the SIGMA website (https://www.sigmaaldrich.com/life-science/stem-cell-biology/system-cell-protocols.html). reference). For the culture, a culture medium (ESC culture medium) having the following composition was used.
1 mM sodium pyruvate (manufactured by Gibco, catalog number: 11360-070), 1x non-essential amino acid (manufactured by Gibco, catalog number 11140-050), 100 μM 2-mercaptoethanol (manufactured by SIGMA, CAS number: 60-24-) 2), 1000 units/mL leukemia inhibitory factor (LIF, ESGRO (registered trademark), manufactured by Gibco, catalog number: ESG1107), 15% knockout serum substitute (KSR, manufactured by Thermo Fisher Scientific, catalog number: 10828028), and Glasgow minimum essential medium (GMEM medium, Sigma, product number: G5154-500ML) containing 1% fetal bovine serum (FBS, Hyclone). Furthermore, in order to maintain and culture these cells, the surface of a 100 mm culture dish (manufactured by Falcon) was coated with gelatin derived from bovine epidermis (manufactured by Sigma, product number: G9391) and kept at 37° C. for 2 hours or more. The placed one was used.
　ヒト胚性癌（ＥＣ）細胞）（ＮＴ２）は、Ａｍｅｒｉｃａｎ　Ｔｉｓｓｕｅ　Ｃｕｌｔｕｒｅ　Ｃｏｌｌｅｃｔｉｏｎから入手し（ＣＲＬ－１９７３）、１０％ＦＢＳ含有ＤＭＥＭ（ダルベッコ改変イーグル培地）にて培養した。 Human embryonal carcinoma (EC) cells (NT2) were obtained from the American Tissue Culture Collection (CRL-1973) and cultured in DMEM (Dulbecco's modified Eagle medium) containing 10% FBS.
　（分化細胞）
　マウス神経幹細胞（ｍＮＳＣ）は、Ｊｏｈｅ　ＫＫら、Ｇｅｎｅｓ　Ｄｅｖ．１９９６年、１０巻、２４号、３１２９～３１４０ページに記載のとおり、マウス（胎仔１４．５日目）の終脳から調製し、ＮＳＣ培養液［ｂＦＧＦ（１０ｎｇ／ｍｌ）、ＥＧＦ（１０ｎｇ／ｍｌ）、ヘパリン（５μＭ）、ＧｌｕｔａＭＡＸ（Ｇｉｂｃｏ社製）、ペニシリン、及びストレプトマイシンを含むＤＭＥＭ／Ｆ１２培地（Ｓｉｇｍａ社製）］にて培養した。 (Differentiated cells)
Mouse neural stem cells (mNSCs) are described in Johe KK et al., Genes Dev. 1996, Vol. 10, No. 24, pp. 3129-3140, prepared from the telencephalon of mice (fetus 14.5 days), and cultured in NSC medium [bFGF (10 ng/ml), EGF (10 ng/ml). ), heparin (5 μM), GlutaMAX (manufactured by Gibco), penicillin, and streptomycin in DMEM/F12 medium (manufactured by Sigma)].
　マウス骨髄由来ストローマ細胞（ＰＡ６）は、理研バイオリソースセンターから入手し（ＲＢＲＣ－ＲＣＢ１１２７）、１０％ＦＢＳ含有αＭＥＭ（イーグル最小必須培地　α改変型）にて培養した。 Mouse bone marrow-derived stromal cells (PA6) were obtained from Riken BioResource Center (RBRC-RCB1127) and cultured in αMEM containing 10% FBS (Eagle minimum essential medium α modified type).
　マウス筋芽細胞（Ｃ２Ｃ１２）は、理研バイオリソースセンターから入手し（ＲＢＲＣ－ＲＣＢ０９８７）、１０％ＦＢＳ含有ＤＭＥＭにて培養した。 Mouse myoblasts (C2C12) were obtained from Riken BioResource Center (RBRC-RCB0987) and cultured in DMEM containing 10% FBS.
　アストロサイトは、ｍＮＳＣを１０％ＦＢＳ含有ＤＭＥＭ存在下で３日間培養することにより調製、その後、同じ培地で維持した。 Astrocytes were prepared by culturing mNSC in the presence of DMEM containing 10% FBS for 3 days, and then maintained in the same medium.
　（ＭＴＴアッセイ法）
　９６ウェルプレートの各ウェルに、１０００個の各細胞を播種し、ＤＭＳＯのみを終濃度０．１％になるよう添加した培地（２００μＬ／ウェル）にて、又は各ジヒドロオロト酸デヒドロゲナーゼ（ＤＨＯＤＨ）阻害剤を添加した培地（２００μＬ／ウェル）にて、３日間培養した。なお、培地は、上述の各細胞に適した培養液を用いた。 (MTT assay method)
1000 cells were seeded in each well of a 96-well plate and DMSO alone was added to a medium (200 μL/well) to a final concentration of 0.1%, or each dihydroorotic acid dehydrogenase (DHODH) inhibitor The cells were cultured for 3 days in a medium (200 μL/well) supplemented with. The culture medium used was a culture medium suitable for each of the cells described above.
　ＤＨＯＤＨ阻害剤として、Ｂｒｅｑｕｎａｒ（ＢＲＱ，Ｃａｙｍａｎ　Ｃｈｅｍｉｃａｌ，ＣＡＳ番号：９６１８７－５３－０），Ｌｅｆｌｕｎｏｍｉｄｅ（Ｃａｙｍａｎ　Ｃｈｅｍｉｃａｌ，ＣＡＳ番号：７５７０６－１２－６），Ｔｅｒｉｆｌｕｎｏｍｉｄｅ（Ｃａｙｍａｎ　Ｃｈｅｍｉｃａｌ，ＣＡＳ番号：１６３４５１－８１－８）又はＶｉｄｏｆｌｕｄｉｍｕｓ（Ｃａｙｍａｎ，ＣＡＳ番号：７１７８２４－３０－１）を、培地に添加した。各ＤＨＯＤＨ阻害剤の培地への添加濃度は、２００μＭからの２倍希釈系列とした。 As a DHODH inhibitor, Brequnar (BRQ, Cayman Chemical, CAS number: 96187-53-0), Leflunomide (Cayman Chemical, CAS number: 75706-12-6), Teriflunomide (Cayman45:Chamical 81ChemicalChemical number: 81:Chamman: 45-81-Chemical number: 45-81-Chemical number: 16-Chemical-Chemical number) ) Or Vidofludimus (Cayman, CAS number: 717824-30-1) was added to the medium. The concentration of each DHODH inhibitor added to the medium was a 2-fold dilution series starting from 200 μM.
　また、バックグランドとして、培地のみを入れたウェルを用意し、さらにコントロールとして、ＴｒｉｔｏｎＸ－１００（終濃度０．２％）を含む培地を細胞に添加したウェルも用意した。 As a background, a well containing only the medium was prepared, and as a control, a well containing a medium containing Triton X-100 (final concentration 0.2%) was added to the cells.
　そして、各ウェルにＭＴＴ（５ｍｇ／ｍｌ、Ｗａｋｏ社製）５μＬを添加し、細胞を３７℃で２～３時間インキュベートした。次いで、培地を１００μＬのＤＭＳＯに置換して細胞を溶解し、それら細胞溶解液の波長５７０ｎｍにおける吸光度を、マイクロプレートリーダー（Ｂｉｏ－Ｒａｄ社製、Ｂｅｎｃｈｍａｒｋモデル）にて測定した。また、得られた吸光度に基づき、細胞生存率は、以下の式によって算出した。
細胞生存率（％）＝（ＤＨＯＤＨ阻害剤添加ウェルにおける吸光度－バックグランドにおける吸光度）/（コントロールにおける吸光度－バックグランドにおける吸光度）×１００。 Then, 5 μL of MTT (5 mg/ml, manufactured by Wako) was added to each well, and the cells were incubated at 37° C. for 2 to 3 hours. Next, the medium was replaced with 100 μL of DMSO to lyse the cells, and the absorbance of the cell lysate at a wavelength of 570 nm was measured with a microplate reader (Bio-Rad, Benchmark model). The cell viability was calculated by the following formula based on the obtained absorbance.
Cell viability (%)=(absorbance in DHODH inhibitor-added well-absorbance in background)/(absorbance in control-absorbance in background)×100.
　（ヌクレオシド中和実験）
　ＥＳ細胞又はｉＰＳ細胞を、ＢＲＱ（１０μＭ）の存在下、又はＢＲＱ（１０μＭ）及び様々な濃度のリボヌクレオシド（～５００μＭ、全てＳｉｇｍａ社製）の存在下にて、３日間インキュベートし、上述のＭＴＴアッセイにより細胞生存率を分析した。 (Nucleoside neutralization experiment)
ES cells or iPS cells were incubated for 3 days in the presence of BRQ (10 μM) or in the presence of BRQ (10 μM) and various concentrations of ribonucleosides (up to 500 μM, all manufactured by Sigma), and the MTT described above was added. Cell viability was analyzed by assay.
　（免疫染色）
　細胞を、Ｋｏｎｄｏら、Ｇｅｎｅｓ　Ｄｅｖ．２００４年、１８巻、２３号、２９６３～７２ページに記載の方法にて、固定し、免疫染色を施した。具体的には先ず、免疫染色に際して、細胞を、コーティングした８ウェルチャンバースライド（ガラス）上に播種し、１０μＭ　ＢＲＱの存在下で２４時間培養した。なお、ｍＮＳＣの培養には、ＰＤＬ＋フィブロネクチンコートを施したものを用いた（Ｊｏｈｅ　ＫＫら、Ｇｅｎｅｓ　Ｄｅｖ．１９９６年、１０巻、２４号、３１２９～３１４０ページ　参照）。ＥＳ細胞及びｉＰＳ細胞の培養にはゼラチンコートを施したものを用いた。 (Immunostaining)
Cells were cultured in Kondo et al., Genes Dev. It was fixed and immunostained by the method described in 2004, Vol. 18, No. 23, pp. 2963-72. Specifically, first, for immunostaining, cells were seeded on a coated 8-well chamber slide (glass) and cultured in the presence of 10 μM BRQ for 24 hours. For mNSC culture, PDL+fibronectin-coated cells were used (see Joe KK et al., Genes Dev. 1996, 10, 24, 3129-3140). Gelatin-coated cells were used for the culture of ES cells and iPS cells.
　そして、免疫染色においては先ず、ＰＢＳにて細胞を１回洗浄した後、４％パラフォルムアルデヒドを加えて室温で１０分間インキュベートすることにより固定した。０．３％ＴｒｉｔｏｎＸ１００含有ＰＢＳで置換して室温で５分間インキュベートした後、０．３％ＴｒｉｔｏｎＸ１００及び１０％ＦＣＳ含有ＰＢＳ（ブロッキング溶液）に置換して室温で３０分間インキュベートした。ブロッキング溶液で希釈した１次抗体溶液に置換し、室温で２時間インキュベートした。ＰＢＳで３回洗浄した後、ブロッキング溶液で希釈した２次抗体及びＤＡＰＩ溶液を加えて、室温で２時間インキュベートした。ＰＢＳで３回洗浄した後、退色防止剤（ＤＡＫＯ社製、コード番号：Ｓ３０２３）で封入し、顕微鏡観察を行った。なお、ＤＡＰＩ溶液（１μｇ／ｍＬ、Ｄｏｊｉｎｄｏ社製、製品コード：Ｄ５２３）による対比染色によって、細胞核を可視化した。また、蛍光画像は、ＡｘｉｏＩｍａｇｅｒ　Ｍ１顕微鏡（Ｃａｒｌ　Ｚｅｉｓｓ社製）にて取得した。 Then, in the immunostaining, first, the cells were washed once with PBS, added with 4% paraformaldehyde, and incubated at room temperature for 10 minutes for fixation. After replacing with PBS containing 0.3% Triton X100 and incubating at room temperature for 5 minutes, it was replaced with PBS containing 0.3% Triton X100 and 10% FCS (blocking solution) and incubated at room temperature for 30 minutes. The solution was replaced with a primary antibody solution diluted with a blocking solution, and incubated at room temperature for 2 hours. After washing three times with PBS, the secondary antibody diluted with the blocking solution and the DAPI solution were added, and the mixture was incubated at room temperature for 2 hours. After washing three times with PBS, the cells were encapsulated with an anti-fading agent (manufactured by DAKO, code number: S3023) and observed under a microscope. The cell nuclei were visualized by counterstaining with a DAPI solution (1 μg/mL, manufactured by Dojindo, product code: D523). The fluorescence image was acquired with an AxioImager M1 microscope (Carl Zeiss).
　上記培養細胞に対する免疫染色において、抗原の検出には以下の抗体を用いた。
一次抗体：
抗Ｎｅｓｔｉｎマウスモノクローナル抗体（ＢＤ社製，クローン　Ｒａｔ４０１、１：２００に希釈して使用）、
抗Ｎａｎｏｇウサギポリクローナル抗体（Ｗａｋｏ　ｐｕｒｅ　ｃｈｅｍｉｃａｌ社製、コード番号：０１８－２７５２１、１：２００に希釈して使用）、
抗Ｓｏｘ２ウサギポリクローナル抗体（ＳｔｅｍＣｅｌｌ　Ｔｅｃｈｎｏｌｏｇｙ社製、１：５００に希釈して使用）、
抗Ｏｃｔ４マウスモノクローナル抗体（Ｃｅｌｌ　Ｓｉｇｎａｌｉｎｇ　Ｔｅｃｈ社製、１：２００に希釈して使用）、
抗ＧＦＰラットモノクローナル抗体（ナカライテスク社製、１：５００に希釈して使用）、
抗Ｋｉ６７ウサギモノクローナル抗体（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社製、１：１００に希釈して使用）、
抗活性型Ｃａｓｐａｓｅ３ウサギポリクローナル抗体（ＳｔｅｍＣｅｌｌ　Ｔｅｃｈｎｏｌｏｇｙ社製、１：１０００に希釈して使用）。
二次抗体：
Ａｌｅｘａ４８８標識抗マウスＩｇＧロバポリクローナル抗体（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社製、カタログ番号：Ｒ３７１２０、１：５００に希釈して使用）、
Ａｌｅｘａ４８８標識抗ウサギＩｇＧロバポリクローナル抗体（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社製、カタログ番号：Ａ－１１００８、１：５００に希釈して使用）、
Ａｌｅｘａ５９４標識抗ウサギＩｇＧロバポリクローナル抗体（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社製、カタログ番号：Ｒ３７１１９、１：５００に希釈して使用）、
Ａｌｅｘａ５９４標識抗マウスＩｇＧロバポリクローナル抗体（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社製、カタログ番号：Ｒ３７１１５、１：５００に希釈して使用）、
Ａｌｅｘａ４８８標識抗ラットＩｇＧヤギ抗体（Ｊａｃｋｓｏｎ　ＩｍｍｕｎｏＲｅｓｅａｒｃｈ社製、１：５００に希釈して使用）。 In the immunostaining of the above-mentioned cultured cells, the following antibodies were used to detect the antigen.
Primary antibody:
Anti-Nestin mouse monoclonal antibody (BD, clone Rat401, diluted 1:200 for use),
Anti-Nanog rabbit polyclonal antibody (manufactured by Wako pure chemical, code number: 018-27521, used by diluting 1:200),
Anti-Sox2 rabbit polyclonal antibody (manufactured by StemCell Technology, diluted 1:500 for use),
Anti-Oct4 mouse monoclonal antibody (Cell Signaling Tech, diluted 1:200 for use),
Anti-GFP rat monoclonal antibody (manufactured by Nacalai Tesque, Inc., diluted 1:500),
Anti-Ki67 rabbit monoclonal antibody (Thermo Fisher Scientific, manufactured at a dilution of 1:100),
Anti-active Caspase3 rabbit polyclonal antibody (manufactured by StemCell Technology, diluted 1:1000 and used).
Secondary antibody:
Alexa488-labeled anti-mouse IgG donkey polyclonal antibody (Thermo Fisher Scientific, catalog number: R37120, diluted 1:500 for use),
Alexa488-labeled anti-rabbit IgG donkey polyclonal antibody (Thermo Fisher Scientific, catalog number: A-11008, diluted 1:500 for use),
Alexa 594-labeled anti-rabbit IgG donkey polyclonal antibody (Thermo Fisher Scientific, catalog number: R37119, diluted 1:500 for use),
Alexa 594-labeled anti-mouse IgG donkey polyclonal antibody (Thermo Fisher Scientific, catalog number: R37115, diluted 1:500 for use),
Alexa488 labeled anti-rat IgG goat antibody (manufactured by Jackson ImmunoResearch, diluted 1:500 and used).
　腫瘍については、厚さ１０μｍの切片を調製し、Ｋｏｎｄｏ　Ｔ，及びＲａｆｆ　Ｍ．、ＥＭＢＯ　Ｊ．２０００；１９（９）：１９９８－２００７、並びにＴａｋａｎａｇａ　Ｈ，ら、Ｓｔｅｍ　Ｃｅｌｌｓ．２００９；２７（１）：１６５－１７４．に記載の方法にて免疫染色を行った。当該免疫染色には、以下の抗体を用いた。
一次抗体：
抗Ｏｃｔ４マウスモノクローナル抗体（Ｃｅｌｌ　Ｓｉｇｎａｌｉｎｇ　Ｔｅｃｈ社製、１：２００に希釈して使用）、
抗Ｎａｎｏｇウサギポリクローナル抗体（Ｗａｋｏ　Ｐｕｒｅ　Ｃｈｅｍｉｃａｌ社製、１：２００に希釈して使用）、
抗Ｋｉ６７ウサギモノクローナル抗体（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社製、１：１００に希釈して使用）、
抗βIIIチューブリンマウスモノクローナル抗体（Ｓｉｇｍａ社製、１：４００に希釈して使用）、
抗平滑筋アクチンウサギポリクローナル抗体（Ｐｒｏｔｅｉｎｔｅｃｈ社製、ＳＭＡ、１：２００に希釈して使用）、
抗αフェトプロテインウサギポリクローナル抗体（Ｐｒｏｔｅｉｎｔｅｃｈ社製、ＡＦＰ、１：１００に希釈して使用）。
二次抗体：
Ａｌｅｘａ５９４標識抗マウスＩｇＧロバポリクローナル抗体（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社製、カタログ番号：Ｒ３７１１５、１：５００に希釈して使用）、
Ａｌｅｘａ４８８標識抗ウサギＩｇＧヤギ抗体（Ｊａｃｋｓｏｎ　ＩｍｍｕｎｏＲｅｓｅａｒｃｈ社製、１：５００に希釈して使用）。 For tumors, 10 μm thick sections were prepared and examined by Kondo T, and Raff M. et al. EMBO J.; 2000;19(9):1998-2007, and Takanaga H, et al., Stem Cells. 2009;27(1):165-174. Immunostaining was performed by the method described in 1. The following antibodies were used for the immunostaining.
Primary antibody:
Anti-Oct4 mouse monoclonal antibody (Cell Signaling Tech, diluted 1:200 for use),
Anti-Nanog rabbit polyclonal antibody (manufactured by Wako Pure Chemical, diluted 1:200 for use),
Anti-Ki67 rabbit monoclonal antibody (Thermo Fisher Scientific, manufactured at a dilution of 1:100),
Anti-βIII tubulin mouse monoclonal antibody (Sigma, diluted 1:400 for use),
Anti-smooth muscle actin rabbit polyclonal antibody (manufactured by Proteintech, SMA, diluted 1:200 for use),
Anti-alpha fetoprotein rabbit polyclonal antibody (Proteintech, AFP, diluted 1:100 for use).
Secondary antibody:
Alexa 594-labeled anti-mouse IgG donkey polyclonal antibody (Thermo Fisher Scientific, catalog number: R37115, diluted 1:500 for use),
Alexa488-labeled anti-rabbit IgG goat antibody (Jackson ImmunoResearch, 1:500 dilution).
　（混合培養）
　マウスＮＳＣと、マウスＥＳ細胞又はマウスｉＰＳ細胞とを、各々２０００個ずつ播種し、１μＭ又は１０μＭのＢＲＱ存在下、ＥＳＣ培養液にて３日間培養した。そして、上述の免疫染色にて分析した。 (Mixed culture)
2000 mouse NSCs and mouse ES cells or mouse iPS cells were seeded, and cultured in the ESC culture medium for 3 days in the presence of 1 μM or 10 μM BRQ. And it analyzed by the above-mentioned immunostaining.
　（定量ＲＴ－ＰＣＲ）
　マウスＥＳ細胞及びマウスｉＰＳ細胞を、上述のウシ表皮由来のゼラチンにてコーティングした１００ｍｍ培養ディッシュ及びＥＳＣ培養液にて、ＢＲＱ存在下又は非存在下培養した後、これら細胞における、Ｓｏｘ２、Ｏｃｔ４及びＮａｎｏｇの遺伝子発現量を、以下に示す方法にて測定した。 (Quantitative RT-PCR)
Mouse ES cells and mouse iPS cells were cultured in the above-described 100 mm culture dish coated with bovine epidermis-derived gelatin and ESC culture medium in the presence or absence of BRQ, and then Sox2, Oct4, and Nanog in these cells were cultured. The gene expression level was measured by the following method.
　先ず、細胞からＲＮｅａｓｙキット（Ｑｉａｇｅｎ社製）を用いてトータルＲＮＡを調製し、トランスクリプターファーストストランドｃＤＮＡ合成キット（Ｒｏｃｈｅ　Ａｐｐｌｉｅｄ　Ｓｃｉｅｎｃｅ社製）を用いてｃＤＮＡを合成した。 First, total RNA was prepared from cells using an RNeasy kit (manufactured by Qiagen), and cDNA was synthesized using a transcript first-strand cDNA synthesis kit (manufactured by Roche Applied Science).
　そして、当該ｃＤＮＡを鋳型とし、サンダーバード　サイバー　ｑＰＣＲミックス（ＴＯＹＯＢＯ社製）及びステップワンプラス（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社製）を用い、リアルタイムＰＣＲを行なった。反応条件は、９５℃１５秒、６０℃３０秒を４０サイクルとし、以下に示すオリゴヌクレオチドプライマーを使用し、各標的遺伝子を増幅した。
Ｓｏｘ２遺伝子：
フォワードプライマー　５’－ＴＧＡＡＧＡＡＧＧＡＴＡＡＧＴＡＣＡＣＧＣＴ－３’（配列番号：１）、
リバースプライマー　５’－ＴＣＣＴＧＣＡＴＣＡＴＧＣＴＧＴＡＧＣＴＧ－３’（配列番号：２）、
ｏｃｔ４遺伝子：
フォワードプライマー　５’－ＣＴＧＡＡＧＣＡＧＡＡＧＡＧＧＡＴＣＡＣＣ－３’（配列番号：３）、
リバースプライマー　５’－ＣＣＧＣＡＧＣＴＴＡＣＡＣＡＴＧＴＴＣＴＴ－３’（配列番号：４）、
ｎａｎｏｇ遺伝子：
フォワードプライマー　５’－ＣＴＧＡＴＴＣＴＴＣＴＡＣＣＡＧＴＣＣＣＡＡ－３’（配列番号：５）、
リバースプライマー　５’－ＡＧＡＧＴＴＣＴＴＧＣＡＴＣＴＧＣＴＧＧＡ－３’（配列番号：６）、
１８ＳリボゾームＲＮＡ遺伝子：
フォワードプライマー　５’－ＣＧＧＡＣＡＧＧＡＴＴＧＡＣＡＧＡＴＴＧ－３’（配列番号：７）、
リバースプライマー　５’－ＣＡＡＡＴＣＧＣＴＣＣＡＣＣＡＡＣＴＡＡ－３’（配列番号：８）。
なお、このようにして検出された各標的遺伝子の発現量は、１８ＳリボゾームＲＮＡの発量をもとに相対定量法（ｄｅｌｔａ　ｄｅｌｔａ　Ｃｔ法）により正規化した。 Then, using the cDNA as a template, real-time PCR was performed using Thunderbird Cyber qPCR mix (manufactured by TOYOBO) and Step One Plus (manufactured by Thermo Fisher Scientific). The reaction conditions were 95° C. for 15 seconds and 60° C. for 30 seconds for 40 cycles, and each target gene was amplified using the oligonucleotide primers shown below.
Sox2 gene:
Forward primer 5'-TGAAGAAGGAATAAGTACACCGCT-3' (SEQ ID NO: 1),
Reverse primer 5'-TCCTGCATCATGCTGTAGCTG-3' (SEQ ID NO: 2),
oct4 gene:
Forward primer 5′-CTGAAGCAGAAGAGGATCACC-3′ (SEQ ID NO: 3),
Reverse primer 5'-CCGCAGCTTACACATGTTCTT-3' (SEQ ID NO: 4),
nanog gene:
Forward primer 5'-CTGATTCTTCTACCAGTCCCCAA-3' (SEQ ID NO: 5),
Reverse primer 5'-AGAGTTCTTGCATCTGCTGGA-3' (SEQ ID NO: 6),
18S ribosomal RNA gene:
Forward primer 5′-CGGACAGGATTGACAGATTG-3′ (SEQ ID NO: 7),
Reverse primer 5'-CAAATCGCTCCACCAACTAA-3' (SEQ ID NO: 8).
The expression level of each target gene thus detected was normalized by the relative quantification method (delta delta Ct method) based on the amount of 18S ribosomal RNA generated.
　（ベクター）
　ベクターは、Ｎｉｓｈｉｄｅ　Ｋ，ら、ＰＬｏＳ　ＯＮＥ．２００９；４（８）：ｅ６８６９．及びＴａｋａｎａｇａ　Ｈ，ら、Ｓｔｅｍ　Ｃｅｌｌｓ．２００９；２７（１）：１６５－１７４．の記載に沿って構築した。 (vector)
Vectors are based on Nishide K, et al., PLoS ONE. 2009; 4(8):e6869. And Takanaga H, et al., Stem Cells. 2009;27(1):165-174. It was constructed according to the description.
　具体的には先ず、マウスＤＨＯＤＨの全長ｃＤＮＡは、ＫＯＤ　Ｐｌｕｓ－Ｖｅｒ．２ポリメラーゼ（東洋紡株式会社製）を用い、その使用説明書に沿って、マウスＮＳＣ由来ｃＤＮＡから増幅した。当該増幅には、５’プライマー（５’－ＡＧＡＡＴＴＣＡＡＴＧＧＧＧＧＡＡＣＡ－３’　（配列番号：９））及び３’プライマー（５’－ＴＴＧＧＡＴＴＣＴＣＴＧＣＧＧＴＣ－３’（配列番号：１０））を用いた。増幅したｃＤＮＡを、ｐ３ｘＦＬＡＧ　ＣＭＶ１０ベクターに挿入し、ｐ３ｘＦＬＡＧ　ＣＭＶ１０－ｍＤＨＯＤＨを調製した。 Specifically, first, the full-length cDNA of mouse DHODH is KOD Plus-Ver. 2 polymerase (manufactured by Toyobo Co., Ltd.) was used and amplified from mouse NSC-derived cDNA according to the instruction manual. A 5'primer (5'-AGAATTCAATGGGGGAACA-3' (SEQ ID NO: 9)) and a 3'primer (5'-TTGGATTCTCTGCCGGTC-3' (SEQ ID NO: 10)) were used for the amplification. The amplified cDNA was inserted into the p3xFLAG CMV10 vector to prepare p3xFLAG CMV10-mDHODH.
　また、マウスＤＨＯＤＨをノックダウンするために、ＩｎｖｉｖｏＧｅｎのｓｉＲＮ　Ｗｉｚａｒｄソフトウェア（ｈｔｔｐ：／／ｗｗｗ．ｓｉｒｎａｗｉｚａｒｄ．ｃｏｍ／）を用い、当該遺伝子を標的とする３つのショートヘアピン（ｓｈ）配列を選択した。これらｓｈ配列をｐｓｉＲＮＡ－ｈ７ＳＫｈｙｇｒｏ　Ｇ１発現ベクター（ＩｎｖｉｖｏＧｅｎ社製）に各々挿入し、ｐｓｉＲＮＡ－ｈ７ＳＫｈｙｇｒｏ－ｍＤＨＯＤＨｓｈ１－３を調製した。これらベクターによるノックダウン効率はウェスタンブロッティングにより分析した（図１４　参照）。その結果、高い効果を示したＤＨＯＤＨ　ｓｈ１及びｓｈ３をノックダウン実験に用いた。 To knockdown mouse DHODH, we used invivoGen's siRN Wizard software (http://www.sirnawizard.com/) to select three short hairpin (sh) sequences targeting the gene. These sh sequences were inserted into psiRNA-h7SKhygro G1 expression vector (manufactured by InvivoGen) to prepare psiRNA-h7SKhygro-mDHODHsh1-3. The knockdown efficiency of these vectors was analyzed by Western blotting (see Fig. 14). As a result, DHODH sh1 and sh3, which showed a high effect, were used in the knockdown experiment.
　マウスＤＨＯＤＨを対象とするｓｈ１及びのｓｈ３の標的配列は、それぞれ５’－ＧＧＣＴＡＧＣＴＧＴＣｔＣＴＣＴＣＴ－３’（配列番号：１１）及び５’－ＧＧＡＡＧＣＴＧＴＧＴＣＴＣＴＣｔＡ－３’（配列番号：１２）である。コントロールとして用いたｓｈ（ｅｇｆｐ）の標的配列は５’－ＧＣＡＡＧＣＴＧＡＣＣＣＧＴＧＴＴＣＡ－３’（配列番号：１３）である。 The target sequences of sh1 and sh3 targeting mouse DHODH are 5'-GGCTAGCTGTCtCTCTCT-3' (SEQ ID NO: 11) and 5'-GGAAGCTGTGTCTCTCA-3' (SEQ ID NO: 12), respectively. The target sequence of sh(egfp) used as a control is 5'-GCAAGCTGACCCGTGTTTCA-3' (SEQ ID NO: 13).
　クローンニングしたｃＤＮＡの塩基配列は、ＢｉｇＤｙｅターミネーターキットバージョン３．１（Ａｐｐｌｉｅｄ　Ｂｉｏｓｙｓｔｅｍｓ社製）及びＡＢＩシーケンサーモデル３１３０ｘｌ（Ａｐｐｌｉｅｄ　Ｂｉｏｓｙｓｔｅｍｓ社製）を用いて確認した。 The nucleotide sequence of the cloned cDNA was confirmed using BigDye terminator kit version 3.1 (manufactured by Applied Biosystems) and ABI sequencer model 3130xl (manufactured by Applied Biosystems).
　また、ベクターは、リポフェクタミン３０００（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社製）を用い、その使用説明書の記載に沿って、細胞に導入した。 Also, Lipofectamine 3000 (Thermo Fisher Scientific Co., Ltd.) was used as a vector, and the vector was introduced into cells according to the instruction manual.
　（テラトーマ形成）
　多能性幹細胞を、ＢＲＱの存在下又は非存在下で２日間培養した。生存細胞（１ｘ１０６）を、５０μｌのマトリゲル（ＢＤ　Ｂｉｏｓｃｉｅｎｃｅｓ社製）に懸濁し、１０％ペントバルビタールで麻酔した、５～８週齢のＮＯＤ／ＳＣＩＤマウスの臀部に皮下注射した。そして、注射してから４週後に、マウスを犠牲死させ、Ｔｓｕｋａｍｏｔｏ　Ｙ，ら、Ｓｔｅｍ　Ｃｅｌｌｓ．２０１６；３４（８）：２０１６－２０２５．に記載の方法に沿って、腫瘍を摘出して撮影し、それらのサイズを測定した。また得られた測定値に基づき、腫瘍の体積は、以下の式によって算出した。
腫瘍の体積（ｃｍ３）＝長径×短径×短径×１／２。 (Teratoma formation)
Pluripotent stem cells were cultured for 2 days in the presence or absence of BRQ. Viable cells (1×10 6 ) were suspended in 50 μl of Matrigel (manufactured by BD Biosciences) and anesthetized with 10% pentobarbital, and subcutaneously injected into the buttocks of 5-8 week old NOD/SCID mice. Then, 4 weeks after the injection, the mice were sacrificed, and Tsukamoto Y, et al., Stem Cells. 2016;34(8):2016-2025. Tumors were excised, photographed, and their sizes were measured according to the method described in 1. Further, the volume of the tumor was calculated by the following formula based on the obtained measured value.
Tumor volume (cm 3) = long diameter × short diameter × short diameter × 1/2.
　また、ＢＲＱのテラトーマ形成抑制活性を調べるために、皮下腫瘍を有するマウスの腹腔内に、２００μｌの生理食塩水、又は２５ｍｇ／ｋｇブレキナーナトリウム塩（ＢＲＱ、ＴＯＣＲＩＳ社製）ＰＢＳ溶液　２００μｌを、毎日投与した。腹腔内投与の５日後に、マウスを犠牲死させ、Ｔｓｕｋａｍｏｔｏ　Ｙ，ら、Ｓｔｅｍ　Ｃｅｌｌｓ．２０１６；３４（８）：２０１６－２０２５．に記載の方法に沿って、腫瘍を摘出して撮影し、病理学的分析に供した。 Further, in order to examine the teratoma formation inhibitory activity of BRQ, 200 μl of physiological saline or 25 μg/kg of Brekiner sodium salt (BRQ, TOCRIS) PBS solution 200 μl was intraperitoneally injected into the abdominal cavity of mice with subcutaneous tumor every day. Was administered. Five days after the intraperitoneal administration, the mice were sacrificed, and Tsukamoto Y, et al., Stem Cells. 2016;34(8):2016-2025. The tumor was excised, photographed and subjected to pathological analysis according to the method described in 1.
　（ウェスタンブロッティング）
　ウェスタンブロッティングは、Ｔａｋａｎａｇａ　Ｈ，ら、Ｓｔｅｍ　Ｃｅｌｌｓ．２００９；２７（１）：１６５－１７４．に記載の方法に沿って行なった。また、ウェスタンブロッティング及び免疫沈降には、一次抗体として、抗ＦＬＡＧ　Ｍ２マウス抗体（ＳＩＧＭＡ社製、１０μ／ｍｌ）及び抗ＧＡＰＤＨ抗体（Ｐｒｏｔｅｉｎｔｅｃｈ社製、１：５０００に希釈して使用）を用い、二次抗体として、西洋ワサビペルオキシダーゼ標識抗マウスＩｇＧ抗体（Ｓａｎｔａ　Ｃｒｕｚ社製、１：５０００に希釈して使用）を用いた。 (Western blotting)
Western blotting is described by Takanaga H, et al., Stem Cells. 2009;27(1):165-174. Was carried out according to the method described in. For Western blotting and immunoprecipitation, an anti-FLAG M2 mouse antibody (manufactured by SIGMA, 10 μ/ml) and an anti-GAPDH antibody (manufactured by Proteintech, diluted 1:5000 and used) were used as primary antibodies. As a secondary antibody, a horseradish peroxidase-labeled anti-mouse IgG antibody (manufactured by Santa Cruz, diluted 1:5000 for use) was used.
　（統計解析）
　上記にて得られた細胞生存率及び遺伝子発現量等における２群間の比較は、ｓｔｕｄｅｎｔ　ｔ－検定にて解析した。また、カプラン－マイヤー曲線は、無調整のイベント発生迄の時間（ｕｎａｄｊｕｓｔｅｄ　ｔｉｍｅ－ｔｏ－ｅｖｅｎｔ　ｖａｒｉａｂｌｅｓ）を推定するために用いた。P値が０．０５未満（両側検定）である場合、有意であると判断した。 (Statistical analysis)
The comparison between the two groups in the cell viability and the gene expression level obtained above was analyzed by the student t-test. The Kaplan-Meier curve was also used to estimate the unadjusted time-to-event variables. A P value of less than 0.05 (two-sided test) was considered significant.
　（実施例１）　多能性幹細胞に対するＤＨＯＤＨ阻害剤の細胞傷害活性についての検討
　マウスＥＳ細胞及びマウスｉＰＳ細胞を、ＤＨＯＤＨ阻害剤（ＢＲＱ、Ｌｅｆｌｕｎｏｍｉｄｅ、Ｔｅｒｉｆｌｕｎｏｍｉｄｅ又はＶｉｄｅｆｌｕｄｉｍｕｓ）の存在下で各々３日間培養し、生存率をＭＴＴアッセイにより検討した。 (Example 1) Examination on cytotoxic activity of DHODH inhibitor against pluripotent stem cells Mouse ES cells and mouse iPS cells were cultured for 3 days in the presence of DHODH inhibitors (BRQ, Leflunomide, Teriflunomide or Videfludimus), respectively. , The survival rate was examined by the MTT assay.
　その結果、図１及び２に示すとおり、試験した４種のＤＨＯＤＨ阻害剤は全て、両多能性幹細胞に対する細胞傷害活性を有していることが明らかになった。特に、ＢＲＱに関しては、低濃度（１０μＭ～）でも、多能性幹細胞に対する有意な細胞傷害活性が認められた。なお、ＢＲＱ、Ｌｅｆｌｕｎｏｍｉｄｅ、Ｔｅｒｉｆｌｕｎｏｍｉｄｅ及びＶｉｄｏｆｌｕｄｉｍｕｓのヒト由来のＤＨＯＤＨに対するＩＣ５０は各々、６～２０ｎＭ、９８μＭ、１μＭ及び１３４ｎＭである。そのため、ＤＨＯＤＨ阻害剤間における多能性幹細胞に対する細胞傷害活性の差は、阻害活性の差に起因しているように見受けられる。 As a result, as shown in FIGS. 1 and 2, it was revealed that all of the four types of DHODH inhibitors tested had cytotoxic activity against pluripotent stem cells. In particular, regarding BRQ, significant cytotoxic activity against pluripotent stem cells was observed even at low concentrations (10 μM-). The IC 50 of BRQ, Leflunomide, Teriflunomide, and Vidofludimus for human-derived DHODH are 6 to 20 nM, 98 μM, 1 μM, and 134 nM, respectively. Therefore, the difference in cytotoxic activity against pluripotent stem cells between DHODH inhibitors seems to be due to the difference in inhibitory activity.
　（実施例２）　体性幹細胞等に対するＤＨＯＤＨ阻害剤の細胞傷害活性についての検討
　マウス正常神経幹細胞（ｍＮＳＣ）、５％ウシ胎仔血清存在下で培養したｍＮＳＣ（ｍＮＳＣ＋５％ＦＣＳ）、マウスアストロサイトを、１０μＭ　ＢＲＱ存在下で３日間培養し、生存率をＭＴＴアッセイにより検討した。その結果、図３に示すとおり、神経幹細胞及び神経細胞（分化細胞）はＢＲＱに対して低感受性であることが明らかになった。 (Example 2) Examination on cytotoxic activity of DHODH inhibitor on somatic stem cells, etc. Mouse normal neural stem cells (mNSC), mNSCs cultured in the presence of 5% fetal calf serum (mNSC+5% FCS), mouse astrocytes, After culturing for 3 days in the presence of 10 μM BRQ, the survival rate was examined by MTT assay. As a result, as shown in FIG. 3, it was revealed that neural stem cells and neural cells (differentiated cells) have low sensitivity to BRQ.
　また、他の多能性幹細胞（ＮＴ２：ヒト胚性癌（ＥＣ）細胞）及び他の体性幹細胞（ＰＡ６：マウス骨髄由来ストローマ細胞、Ｃ２Ｃ１２：マウス筋芽細胞）についても、１０μＭ　ＢＲＱ存在下で３日間培養し、生存率をＭＴＴアッセイにより検討した。その結果、図４に示すとおり、ＢＲＱは多能性幹細胞（ＥＳ細胞、ｉＰＳ細胞及びＥＣ細胞）では顕著な細胞傷害活性を示した。一方、ＥＳ細胞及びｉＰＳ細胞の生存率と、ＰＡ６細胞、Ｃ２Ｃ１２細胞、神経幹細胞及びアストロサイトのそれとの間に統計学的有意差が認められた（全てｐ＜０．０１）。すなわち、ＢＲＱの体性幹細胞及び体細胞に対する有意な細胞傷害活性は認められなかった。 In addition, other pluripotent stem cells (NT2: human embryonal carcinoma (EC) cells) and other somatic stem cells (PA6: mouse bone marrow-derived stromal cells, C2C12: mouse myoblasts) in the presence of 10 μM BRQ After culturing for 3 days, the survival rate was examined by MTT assay. As a result, as shown in FIG. 4, BRQ showed remarkable cytotoxic activity in pluripotent stem cells (ES cells, iPS cells and EC cells). On the other hand, a statistically significant difference was observed between the survival rate of ES cells and iPS cells and that of PA6 cells, C2C12 cells, neural stem cells and astrocytes (all p<0.01). That is, no significant cytotoxic activity of BRQ on somatic stem cells and somatic cells was observed.
　（実施例３）　多能性幹細胞に対するＤＨＯＤＨ阻害剤の細胞特異的傷害活性についての検証
　多能性幹細胞（マウスＥＳ細胞又はマウスｉＰＳ細胞）と、該細胞から分化誘導して得られる細胞と見立てた体性幹細胞（マウス神経幹細胞）とを、様々な濃度のＢＲＱを含むＥＳ細胞培地にて３日間混合培養し、ＮＳＣマーカーであるＮｅｓｔｉｎ及び多能性幹細胞マーカーであるＮａｎｏｇに対する免疫染色を行った。その結果、図５及び６に示すとおり、上述の図１～４に示した結果同様に、１０μＭのＢＲＱ存在下でＥＳ細胞とｉＰＳ細胞は消失したが、神経幹細胞への明らかな細胞傷害は観られなかった。 (Example 3) Verification of cell-specific damaging activity of DHODH inhibitor on pluripotent stem cells Pluripotent stem cells (mouse ES cells or mouse iPS cells) and cells obtained by inducing differentiation of the cells were regarded as the cells. Somatic stem cells (mouse neural stem cells) were mixed and cultured in an ES cell medium containing various concentrations of BRQ for 3 days, and immunostained for NSC Nestin and pluripotent stem cell marker Nanog. As a result, as shown in FIGS. 5 and 6, like the results shown in FIGS. 1 to 4 above, ES cells and iPS cells disappeared in the presence of 10 μM BRQ, but no apparent cytotoxicity to neural stem cells was observed. I couldn't do it.
　（実施例４）　ＤＨＯＤＨ阻害剤の細胞傷害に対する、ヌクレオシド中和活性についての検証
　ピリミジン合成は、新生（ｄｅ　ｎｏｖｏの合成）経路と再利用（サルベージ）経路とによって制御されている。ＤＨＯＤＨは、当該合成経路において律速となる４番目の化学反応を触媒する、ｄｅ　ｎｏｖｏ合成のキー因子である。そこで、ＤＨＯＤＨ阻害剤による多能性幹細胞の除去効果が、サルベージ経路の出発材料であるウリジン等のヌクレオシドを添加することにより、当該効果を解消できるかどうかを調べた。より具体的には、ＢＲＱの存在下、ヌクレオシド（アデノシン、グアノシン、シチジン又はウリジン）を培地に添加することによって、当該阻害剤による多能性幹細胞の細胞傷害活性を中和し得るかを検証した。その結果、図７及び８に示すとおり、ＢＲＱの細胞傷害活性に対し、ピリミジンヌクレオシド（ウリジン、シチジン）は中和活性を示し、特にウリジンは強い中和活性を示すことが明らかになった。 Example 4 Verification of Nucleoside Neutralizing Activity Against Cytotoxicity of DHODH Inhibitors Pyrimidine synthesis is regulated by nascent (de novo synthesis) and salvage (salvage) pathways. DHODH is a key factor in de novo synthesis that catalyzes the fourth rate-limiting chemical reaction in the synthetic pathway. Therefore, it was investigated whether the effect of removing pluripotent stem cells by the DHODH inhibitor can be eliminated by adding a nucleoside such as uridine which is a starting material of the salvage pathway. More specifically, it was verified by adding a nucleoside (adenosine, guanosine, cytidine or uridine) to the medium in the presence of BRQ that the cytotoxic activity of pluripotent stem cells by the inhibitor can be neutralized. .. As a result, as shown in FIGS. 7 and 8, it was revealed that pyrimidine nucleosides (uridine and cytidine) have a neutralizing activity, and particularly uridine has a strong neutralizing activity against the cytotoxic activity of BRQ.
　また、ピリミジン合成経路の産物であるＵＭＰは、ＵＤＰ、ＵＴＰへと変化する。さらに、ＵＴＰを基質としてウリジン二リン酸－Ｎ－アセチルグルコサミン（ＵＤＰ－ＧｌｃＮａｃ）も生合成される。ＵＤＰ－ＧｌｃＮａｃは、Ｏ結合型Ｎ－アセチルグルコサミン転移酵素（ＯＧＴ）の基質となり、細胞内シグナル伝達に広範に関与することが明らかになっている。そこで、ＤＨＯＤＨ阻害剤による多能性幹細胞の細胞傷害活性が、これらＵＭＰからの生成物によっても中和されるかを検証した。その結果、図９及び１０に示すとおり、ＢＲＱの細胞傷害活性に対し、ＵＤＰ及びＵＤＰ－ＧｌｃＮａｃも中和活性を示すことが明らかになった。 Also, UMP, which is a product of the pyrimidine synthesis pathway, changes to UDP and UTP. Furthermore, uridine diphosphate-N-acetylglucosamine (UDP-GlcNac) is also biosynthesized using UTP as a substrate. It has been revealed that UDP-GlcNac serves as a substrate for O-linked N-acetylglucosaminyltransferase (OGT) and is widely involved in intracellular signal transduction. Therefore, it was examined whether the cytotoxic activity of pluripotent stem cells by the DHODH inhibitor was neutralized by the products from these UMPs. As a result, as shown in FIGS. 9 and 10, it was revealed that UDP and UDP-GlcNac also have a neutralizing activity against the cytotoxic activity of BRQ.
　また、ヌクレオチド合成の中間基質であるヌクレオチド二リン酸（ＵＤＰ、ＣＤＰ、ＡＤＰ、ＧＤＰ）についても、ＢＲＱの細胞傷害活性に対する中和活性を評価した。その結果、上記同様に、ＵＤＰにおいて強い中和活性が認められた（図１１　参照）。 Also, for the nucleotide diphosphates (UDP, CDP, ADP, GDP), which are intermediate substrates for nucleotide synthesis, the neutralizing activity against the cytotoxic activity of BRQ was evaluated. As a result, similar to the above, a strong neutralizing activity was recognized in UDP (see FIG. 11).
　以上のことから、ＢＲＱ等のＤＨＯＤＨ阻害剤は、ピリミジン合成経路を阻害することによって、多能性幹細胞に対する細胞傷害活性を奏していることが示唆された。また、その細胞傷害活性は、再利用（サルベージ）経路を活性化することにより抑制できることも示唆された。 From the above, it was suggested that DHODH inhibitors such as BRQ exert cytotoxic activity on pluripotent stem cells by inhibiting the pyrimidine synthesis pathway. It was also suggested that its cytotoxic activity could be suppressed by activating the salvage pathway.
　（実施例５）　ＢＲＱによる多能性幹細胞における細胞周期停止及び細胞死の誘導についての検証
　多能性幹細胞において、ＤＨＯＤＨ阻害剤であるＢＲＱがどのような現象を引き起こしているかを明らかにすべく、多能性幹細胞を、ＢＲＱの存在下又は非存在下にて２日間培養した後、ブロモ－デオキシウリジン（ＢｒｄＵ）の取り込みアッセイとＣａｓｐ３免疫染色を行い、それらの細胞増殖性と細胞死を分析した。その結果、図１２及び１３に示すとおり、ＢＲＱは多能性幹細胞の増殖性を低減し、ＣＡＳＰ３を強く活性化することが明らかとなった。 Example 5 Verification of Induction of Cell Cycle Arrest and Cell Death in Pluripotent Stem Cells by BRQ To clarify what phenomenon the DHODH inhibitor BRQ causes in pluripotent stem cells, After pluripotent stem cells were cultured in the presence or absence of BRQ for 2 days, bromo-deoxyuridine (BrdU) uptake assay and Casp3 immunostaining were performed to analyze their cell proliferation and cell death. .. As a result, as shown in FIGS. 12 and 13, it was revealed that BRQ reduced the proliferation of pluripotent stem cells and strongly activated CASP3.
　また、ＢＲＱ依存性の細胞傷害活性は、ＤＨＯＤＨ活性阻害に完全に依存しているかどうかを評価すべく、２種類のＤＨＯＤＨ特異的ｓｈＲＮＡ（図１４　参照）を用い、ＤＨＯＤＨをノックダウンした。その結果、図１５～１８に示すとおり、多能性幹細胞の増殖性は低減し、ＣＡＳＰ３は強く活性化された。このように、ＢＲＱ処理細胞同様の現象が、ＤＨＯＤＨノックダウンによっても生じたことから、前記ＢＲＱ依存性細胞傷害性は、ＤＨＯＤＨ活性を阻害することによって発揮されたことが示唆された。 Moreover, in order to evaluate whether the BRQ-dependent cytotoxic activity was completely dependent on inhibition of DHODH activity, two types of DHODH-specific shRNA (see FIG. 14) were used to knockdown DHODH. As a result, as shown in FIGS. 15 to 18, proliferation of pluripotent stem cells was reduced and CASP3 was strongly activated. As described above, the phenomenon similar to that of BRQ-treated cells was also caused by DHODH knockdown, suggesting that the BRQ-dependent cytotoxicity was exerted by inhibiting DHODH activity.
　（実施例６）　多能性幹細胞のマーカー分子への、ＤＨＯＤＨ阻害剤による影響についての検証
　多能性幹細胞においては、自己複製能の促進と未分化状態の維持に関わる３種類の転写因子（Ｓｏｘ２、Ｏｃｔ４、Ｎａｎｏｇ）が高いレベルにて発現していることが明らかとなっている。そこで、これら多能性幹細胞のマーカー分子へのＤＨＯＤＨ阻害剤による影響について検証した。 Example 6 Verification of Effect of DHODH Inhibitor on Marker Molecule of Pluripotent Stem Cell In pluripotent stem cells, three transcription factors (Sox2) involved in promotion of self-renewal ability and maintenance of undifferentiated state. , Oct4, Nanog) are expressed at high levels. Therefore, the effect of the DHODH inhibitor on the marker molecules of these pluripotent stem cells was examined.
　具体的には先ず、ＢＲＱ存在下にて２４時間培養した、多能性幹細胞（マウスｉＰＳ細胞及びマウスＥＳ細胞）、並びに体性幹細胞（マウス神経幹細胞）におけるＳｏｘ２の発現を、蛍光免疫染色にて検出した。その結果、図１９及び２０に示すとおり、ＢＲＱ存在下の２４時間培養にて、ＥＳ細胞及びｉＰＳ細胞では早くも細胞増殖阻害、或いは細胞死が観察された。また、両多能性幹細胞の３０％程度においてＳｏｘ２の発現は認められないようになっていた。一方、体性幹細胞においては同様の変化は認められなかった。 Specifically, first, the expression of Sox2 in pluripotent stem cells (mouse iPS cells and mouse ES cells) and somatic stem cells (mouse neural stem cells) cultured for 24 hours in the presence of BRQ was examined by fluorescent immunostaining. Detected. As a result, as shown in FIGS. 19 and 20, cell growth inhibition or cell death was observed in ES cells and iPS cells as early as in 24-hour culture in the presence of BRQ. In addition, expression of Sox2 was not observed in about 30% of the pluripotent stem cells. On the other hand, similar changes were not observed in somatic stem cells.
　次に、ＢＲＱ存在下にて１日又は２日間培養した、多能性幹細胞（マウスｉＰＳ細胞及びマウスＥＳ細胞）におけるＳｏｘ２、Ｏｃｔ４及びＮａｎｏｇの遺伝子発現量を、ＰＣＲにて測定した。その結果、図２１に示すとおり、ＢＲＱにて２日間処理した細胞において、多能性幹細胞のマーカー遺伝子の発現量は総じて減少していることが明らかになった。 Next, the gene expression levels of Sox2, Oct4 and Nanog in pluripotent stem cells (mouse iPS cells and mouse ES cells) cultured in the presence of BRQ for 1 day or 2 days were measured by PCR. As a result, as shown in FIG. 21, it was revealed that the expression level of the pluripotent stem cell marker gene was generally decreased in the cells treated with BRQ for 2 days.
　また、ＢＲＱ存在下にて２日間培養した、多能性幹細胞（マウスｉＰＳ細胞及びマウスＥＳ細胞）におけるＮａｎｏｇの発現を、蛍光免疫染色にて検出した。その結果、図２２に示すとおり、通常、多能性幹細胞の核に局在しているＮａｎｏｇは、ＢＲＱ処理によって細胞質に広がって分布するようになることが明らかになった。 Moreover, the expression of Nanog in pluripotent stem cells (mouse iPS cells and mouse ES cells) cultured for 2 days in the presence of BRQ was detected by fluorescent immunostaining. As a result, as shown in FIG. 22, it was revealed that Nanog, which is normally localized in the nucleus of pluripotent stem cells, becomes spread and distributed in the cytoplasm by BRQ treatment.
　以上の結果から、ＤＨＯＤＨ阻害剤によって、自己複製能の促進と未分化状態の維持に関わる３種類の転写因子（Ｓｏｘ２、Ｏｃｔ４、Ｎａｎｏｇ）の発現量が低減し、また細胞内局在が変化することによって、多能性幹細胞に細胞傷害、ひいては細胞死がもたらされることが示唆される。 From the above results, the expression level of three transcription factors (Sox2, Oct4, Nanog) involved in promotion of self-renewal ability and maintenance of undifferentiated state is decreased by the DHODH inhibitor, and intracellular localization is changed. This suggests that pluripotent stem cells undergo cytotoxicity and eventually cell death.
　（実施例７）　ＤＨＯＤＨ阻害剤によって誘導される、ＣＲＭ１依存的多能性幹細胞マーカー分子の核外輸送
　ＢＲＱ若しくはｐａｎカスパーゼ阻害剤であるＺ－ＶＡＤの存在下又は非存在下において、多能性幹細胞を培養した。ＢＲＱにて処理した後２日目に、多能性幹細胞（ＥＳ細胞及びｉＰＳ細胞）におけるＯｃｔ４陽性細胞及びＮａｎｏｇ陽性細胞の数を測定した。 (Example 7) Nuclear export of CRM1-dependent pluripotent stem cell marker molecule induced by DHODH inhibitor Pluripotent stem cells in the presence or absence of BRQ or pan-caspase inhibitor Z-VAD Were cultured. On the second day after treatment with BRQ, the number of Oct4-positive cells and Nanog-positive cells in pluripotent stem cells (ES cells and iPS cells) was measured.
　その結果、図２３に示すとおり、ＢＲＱにて処理した多能性幹細胞におけるＯｃｔ４陽性細胞及びＮａｎｏｇ陽性細胞の割合は、ＤＭＳＯにて処理したコントロール多能性幹細胞におけるそれらと比較して、有意に減少した（ＢＲＱにて処理したＥＳ細胞における、Ｏｃｔ４陽性細胞：２％、Ｎａｎｏｇ陽性細胞：６％。ＢＲＱにて処理したｉＰＳ細胞における、Ｏｃｔ４陽性細胞：０％、Ｎａｎｏｇ陽性細胞：０％。ＤＭＳＯにて処理したＥＳ細胞における、Ｏｃｔ４陽性細胞：８５％、Ｎａｎｏｇ陽性細胞：８６％。ＤＭＳＯにて処理したｉＰＳ細胞における、Ｏｃｔ４陽性細胞：９４％、Ｎａｎｏｇ陽性細胞：９３％）。 As a result, as shown in FIG. 23, the proportion of Oct4-positive cells and Nanog-positive cells in the BRQ-treated pluripotent stem cells was significantly reduced as compared with those in the DMSO-treated control pluripotent stem cells. (Oct4 positive cells in ES cells treated with BRQ: 2%, Nanog positive cells: 6%. Oct4 positive cells: 0%, Nanog positive cells: 0% in iPS cells treated with BRQ. DMSO Oct4-positive cells: 85%, Nanog-positive cells: 86% in the ES cells treated with, and Oct4-positive cells: 94%, Nanog-positive cells: 93% in the iPS cells treated with DMSO).
　また、Ｚ－ＶＡＤの添加によって、ＢＲＱ依存的細胞死にわずかな遅れを生じたものの、ＢＲＱにて処理した多能性幹細胞における前記Ｎａｎｏｇ陽性細胞及びＯｃｔ４陽性細胞の割合のの減少を回復させることはなかった（ＢＲＱ及びＺ－ＶＡＤにて処理したＥＳ細胞における、Ｏｃｔ４陽性細胞：５％、Ｎａｎｏｇ陽性細胞：２％。ＢＲＱ及びＺ－ＶＡＤにて処理したｉＰＳ細胞における、Ｏｃｔ４陽性細胞：３％、Ｎａｎｏｇ陽性細胞：５％）（図２３　参照）。 Moreover, although the addition of Z-VAD caused a slight delay in BRQ-dependent cell death, it was possible to restore the decrease in the ratio of Nanog-positive cells and Oct4-positive cells in BRQ-treated pluripotent stem cells. Not present (Oct4 positive cells: 5%, Nanog positive cells: 2% in ES cells treated with BRQ and Z-VAD. Oct4 positive cells: 3% in iPS cells treated with BRQ and Z-VAD, Nanog positive cells: 5%) (see FIG. 23).
　また、ＢＲＱ存在下又は非存在下、レプトマイシンＢ（ＬＭＢ）を添加した培地にて、多能性幹細胞を培養し、Ｎａｎｏｇ及びＯｃｔ４の核局在を分析した。なお、ＬＭＢは、Ｅｘｐｏｒｔｉｎ　１としても知られるＣＲＭ１に対する特異的阻害剤である。 Also, pluripotent stem cells were cultured in a medium supplemented with leptomycin B (LMB) in the presence or absence of BRQ, and nuclear localization of Nanog and Oct4 was analyzed. LMB is a specific inhibitor of CRM1 also known as Exportin1.
　その結果、図２４に示すとおり、ＬＭＢは、ＢＲＱにて処理した多能性幹細胞におけるＮａｎｏｇ及びＯｃｔ４の核外輸送を完全に抑制した。このことから、ＢＲＱによるＮａｎｏｇ及びＯｃｔ４の核からの排除は、ＣＲＭ１依存的に生じていたことが示唆される。 As a result, as shown in FIG. 24, LMB completely suppressed the nuclear export of Nanog and Oct4 in BRQ-treated pluripotent stem cells. This suggests that the elimination of Nanog and Oct4 from the nucleus by BRQ occurred in a CRM1-dependent manner.
　（実施例８）　多能性幹細胞の腫瘍形成能に対する、ＤＨＯＤＨ阻害剤の抑制効果についての検証
　ＤＨＯＤＨ阻害剤であるＢＲＱにて前処理した多能性幹細胞の腫瘍形成能について調べた。具体的には先ず、ＤＭＳＯのみ又は１０μＭ　ＢＲＱを添加した培地にて２日間培養した後、生存していた多能性幹細胞をＮＯＤ／ＳＣＩＤマウスの臀部皮下に注入した。そして、移植してから４週間後に、腫瘍を摘出して観察した。 (Example 8) Verification of suppressive effect of DHODH inhibitor on tumorigenicity of pluripotent stem cells The tumorigenicity of pluripotent stem cells pretreated with BRQ which is a DHODH inhibitor was examined. Specifically, first, after culturing in a medium supplemented with DMSO alone or 10 μM BRQ for 2 days, surviving pluripotent stem cells were subcutaneously injected into the buttocks of the NOD/SCID mice. Then, 4 weeks after the transplantation, the tumor was excised and observed.
　その結果、図２５及び２６に示すとおり、ＢＲＱにて前処理した細胞は増殖していなかった。一方、ＤＭＳＯにて処理した細胞においては、腫瘍形成が認められた。 As a result, as shown in FIGS. 25 and 26, the cells pretreated with BRQ were not proliferating. On the other hand, tumor formation was observed in the cells treated with DMSO.
　また、ＢＲＱの抗テラトーマ形成活性を調べるため、先ず、ＮＯＤ／ＳＣＩＤマウスの臀部皮下に多能性幹細胞を移植し、１００ｍｍ３を超えるサイズに達する腫瘍を形成させた。そして、３日毎に１回、ＢＲＱを腹腔内注射し、５回目の腹腔内注射を施してから３日目に、マウスから腫瘍を摘出した。 In order to examine the anti-teratoma formation activity of BRQ, pluripotent stem cells were first transplanted subcutaneously into the buttocks of NOD/SCID mice to form tumors reaching a size of more than 100 mm 3 . Then, BRQ was intraperitoneally injected once every three days, and a tumor was excised from the mouse on day 3 after the fifth intraperitoneal injection.
　図２７に示すとおり、ＢＲＱ投与によって腫瘍の増殖は抑制された。なお、図には示さないが、ＢＲＱを投与したマウスにおいて目に見える副作用は認められなかった。また図２８に示すとおり、ＤＭＳＯ及びＢＲＱにて処理したテラトーマのサイズは各々、ＥＳ細胞由来のもので０．５５ｃｍ３及び０．１２ｃｍ３であり、ｉＰＳ細胞由来のもので、１．３７ｃｍ３及び０．３２ｃｍ３であった。 As shown in FIG. 27, BRQ administration suppressed tumor growth. Although not shown in the figure, no visible side effect was observed in the BRQ-administered mice. As also shown in FIG. 28, teratomas size treated with DMSO and BRQ is each a 0.55 cm 3 and 0.12 cm 3 in those from ES cells, those derived from iPS cells, 1.37Cm 3 and It was 0.32 cm 3 .
　次に、それら腫瘍の切片を調製し、増殖マーカー　Ｋｉ６７、多能性幹細胞マーカー（Ｏｃｔ４及びＮａｎｏｇ）、並びに分化マーカー（ＡＴＦ，βIIIチューブリン及びＳＭＡ）を対象とする免疫標識を施し、観察した。 Next, sections of these tumors were prepared, subjected to immunolabeling with the proliferation marker Ki67, pluripotent stem cell markers (Oct4 and Nanog), and differentiation markers (ATF, βIII tubulin and SMA), and observed.
　その結果、図２９に示すとおり、ＢＲＱ処理腫瘍におけるＫｉ６７陽性増殖細胞は、ＤＭＳＯ処理腫瘍におけるそれらと比較して、有意に減少した。また、図３０に示すとおり、ＢＲＱ処理腫瘍において、Ｏｃｔ４陽性細胞及びＮａｎｏｇ陽性細胞の数は、有意に減少したが、ＤＭＳＯ処理腫瘍において、多能性幹細胞マーカーを発現している細胞は多く認められた。一方、三胚葉における各マーカーであるＡＴＦ，βIIIチューブリン及びＳＭＡを各々発現する細胞を、両腫瘍とも同様に含んでいた。 As a result, as shown in FIG. 29, Ki67-positive proliferating cells in BRQ-treated tumors were significantly reduced as compared with those in DMSO-treated tumors. In addition, as shown in FIG. 30, the number of Oct4-positive cells and Nanog-positive cells was significantly reduced in BRQ-treated tumors, but many cells expressing pluripotent stem cell markers were observed in DMSO-treated tumors. It was On the other hand, both tumors similarly contained cells expressing ATF, βIII tubulin and SMA, which are markers in the three germ layers, respectively.
　したがって、ＤＨＯＤＨ阻害剤は、未分化の多能性幹細胞を特異的に除去することによって、分化細胞及びマウスに対して明白な細胞毒性をもたらすことなく、腫瘍形成を抑制できることが明らかになった。 Therefore, it was revealed that the DHODH inhibitor can suppress tumor formation by specifically removing undifferentiated pluripotent stem cells without causing obvious cytotoxicity to differentiated cells and mice.
　以上説明したように、本発明によれば、分化細胞には有意な細胞傷害をもたらすことなく、未分化の多能性幹細胞を除去することが可能となる。したがって、本発明は、多能性幹細胞から分化誘導した細胞群に残存する未分化の多能性幹細胞を除去することに優れているため、腫瘍化リスクが低減され、また副作用の少ない再生医療等において有用である。 As explained above, according to the present invention, it is possible to remove undifferentiated pluripotent stem cells without causing significant cytotoxicity to the differentiated cells. Therefore, the present invention is excellent in removing undifferentiated pluripotent stem cells remaining in a cell group differentiated from pluripotent stem cells, so that the risk of tumorigenesis is reduced, and regenerative medicine with few side effects, etc. Is useful in.

Claims (8)

  1. 　ジヒドロオロト酸デヒドロゲナーゼ阻害剤を有効成分として含有する、多能性幹細胞から分化誘導した細胞群に残存する未分化の多能性幹細胞を除去するための組成物。 A composition for removing undifferentiated pluripotent stem cells remaining in a cell group differentiated from pluripotent stem cells, which contains a dihydroorotic acid dehydrogenase inhibitor as an active ingredient.
  2. 　前記多能性幹細胞が、胚性幹細胞（ＥＳ細胞）、人工多能性幹細胞（ｉＰＳ細胞）及び胚性腫瘍細胞（ＥＣ細胞）からなる群から選択される少なくとも１の多能性幹細胞である、請求項１に記載の組成物。 The pluripotent stem cells are at least one pluripotent stem cell selected from the group consisting of embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells) and embryonal tumor cells (EC cells), The composition according to claim 1.
  3. 　前記ジヒドロオロト酸デヒドロゲナーゼ阻害剤がブレキナー（ｂｒｅｑｕｉｎａｒ）である、請求項１又は２に記載の組成物。 The composition according to claim 1 or 2, wherein the dihydroorotic acid dehydrogenase inhibitor is brequinar.
  4. 　前記細胞群が、前記多能性幹細胞から分化誘導した体性幹細胞を含む細胞群である、請求項１～３のうちのいずれか一項に記載の組成物。 The composition according to any one of claims 1 to 3, wherein the cell group is a cell group containing somatic stem cells differentiated from the pluripotent stem cells.
  5. 　多能性幹細胞から分化誘導した細胞群とジヒドロオロト酸デヒドロゲナーゼ阻害剤とを接触させる工程を含む、前記細胞群から残存する未分化の多能性幹細胞を除去する方法。 A method for removing the remaining undifferentiated pluripotent stem cells from the cell group, which comprises the step of contacting a cell group differentiated from the pluripotent stem cell with a dihydroorotate dehydrogenase inhibitor.
  6. 　前記多能性幹細胞が、胚性幹細胞（ＥＳ細胞）、人工多能性幹細胞（ｉＰＳ細胞）及び胚性腫瘍細胞（ＥＣ細胞）からなる群から選択される少なくとも１の多能性幹細胞である、請求項５に記載の方法。 The pluripotent stem cells are at least one pluripotent stem cell selected from the group consisting of embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells) and embryonal tumor cells (EC cells), The method according to claim 5.
  7. 　前記ジヒドロオロト酸デヒドロゲナーゼ阻害剤がブレキナー（ｂｒｅｑｕｉｎａｒ）である、請求項５又は６に記載の方法。 The method according to claim 5 or 6, wherein the dihydroorotic acid dehydrogenase inhibitor is brequinar.
  8. 　前記細胞群が、前記多能性幹細胞から分化誘導した体性幹細胞を含む細胞群である、請求項５～７のうちのいずれか一項に記載の方法。 The method according to any one of claims 5 to 7, wherein the cell group is a cell group containing somatic stem cells that are induced to differentiate from the pluripotent stem cells.
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